Biological containment

ABSTRACT

A replicon, in which a nucleotide sequence encoding a cell killing function is regulatably expressed when the replicon is harboured in one type of host cell (primary host cell), so that cells harbouring the replicon are killed under conditions under which the cell killing function is expressed, and the nucleotide sequence encoding the cell killing function is regulatably or constitutively expressed when the replicon is harboured in another type of host cell (secondary host cell), so that cells harbouring the replicon are invariably killed or killed under conditions under which the cell killing function is expressed, may be used in a method of active biological containment of cells under defined environmental conditions. 
     The biological containment principle may be utilized in the industrial production of a biosynthetic product by recombinant DNA techniques, when deliberately releasing a genetically engineered microorganism to the natural environment or in the preparation of a live vaccine. 
     The expression of the cell killing function may be regulated by means of a promoter.

This application is a continuation of application Ser. No. 08/205,824 ,filed Mar. 4, 1994 now abandoned, which is a continuation of applicationSer. No. 07/947,910, filed Sep. 21, 1992 , now abandoned; which iscontinuation of application Ser. No. 07/132,942, filed Nov. 6, 1987 ,now abandoned; which is the national stage of application Ser. No.PCT/DK87/00031 , filed Mar. 25, 1987.

The present invention relates to a method of biologically containing anorganism or a replicon under certain conditions, and a replicon used inthe method, as well as a cell containing said replicon.

TECHNICAL BACKGROUND

The techniques employing the in vitro recombination of DNA moleculeswhich techniques are popularly termed "genetic engineering" have made itpossible to isolate specific genes and express such genes in a varietyof host cells, including host cells in which the genes in question arenot found or expressed in nature. A recombinant DNA molecule typicallyconsists of a vector which is able to replicate autonomously in the hostcells harbouring it or which is integrated into the host cell genome,one or more genes coding for one or more desired biosynthetic productsand DNA sequences required for expression of the gene or genes in thehost cell. The recombinant DNA techniques have become important forindustrial applications such as large-scale fermentation of geneticallyengineered organisms such as bacteria, yeasts or animal cells, toproduce one or more desired biosynthetic-products such as peptidehormones, e.g. insulin and growth hormone, or enzymes such asplasminogen activators; another important area of application is thecontrolled release of genetically engineered microorganisms or virusesinto the environment, for instance bacteria or viruses capable ofkilling larvae of insects which are harmful to certain plants, bacteriadegrading certain pollutants, such as oil, or bacteria which reduce thecold sensitivity of certain crops.

From the earliest stage of development of recombinant DNA techniques inthe 1970s, the scientific community has been highly aware of thepossible biological hazards associated with genetic engineering. As aresult, the National Institutes of Health, Bethesda, USA, proposed a setof "Guidelines for Recombinant DNA Research" which set the standard formost other countries. Since 1978, the Guidelines have been revisedregularly on the basis of accumulated experimental evidence concerningthe possible biological hazards associated with recombinant DNA work.

Despite a tendency to relax the NIH regulations, public opinion remainsgreatly concerned about the possible biological hazards associated withgenetic engineering. Public concern has mainly been directed towardspossible effects of experiments involving the controlled release ofgenetically engineered organisms to the environment. However, in manycountries the large-scale production of biosynthetic substances to beused in connection with therapy and the like has also been questionedwith respect to its safety, especially with respect to the effect of theaccidental release of the recombinant organisms producing suchsubstances from the fermentors to the environment. Therefore, it is notpossible to exploit the industrial potential of genetic engineeringfully, before the safety aspects have been resolved.

In order to avoid or at least reduce the risks associated withexperiments or large-scale applications of genetic engineering, such asthe release of recombinant organisms to the environment, measures havebeen taken to limit the number of such organisms released under ordinaryoperating conditions as well as in the case of certain types of accidentby means of a suitable physical design of laboratories and productionfacilities.

Such measures are termed "physical containment" by which is meant anydesign feature of laboratories or production facilities which isintended to confine the recombinant organisms to a specific,predetermined, restricted area. Different levels of physical containmentare required for different types of recombinant DNA work according toNIH regulations. Thus, work with potential pathogens requires stricterphysical conditions in the laboratory or production facility where thework is carried out.

Physical containment measures are feasible within a laboratory orproduction facility, while no such measures are possible in the case ofapplications involving controlled release of genetically engineeredmicroorganisms to the environment.

Alternatively or concomitantly, the continued survival of accidentallyreleased recombinant organisms or the spread of recombinant DNAmolecules in the environment may be limited by "biological containment".This term is meant to indicate any feature of the host cell or repliconemployed in the production of a specific biosynthetic product oremployed for its ability to bring about a desired event, which featureserves to limit the growth potential of the host cell outside aspecific, restricted environment where specific conditions prevail (inthe following termed "defined environment") and/or any feature of areplicon harboured in the host cell, which feature serves to limit thespread of the replicon (as well as any inserted foreign nucleotidesequence, i.e. a nucleotide sequence which is not naturally related tothe replicon in question) to other organisms than those for which it hasbeen intended, Biological containment may also be obtained through acombination of specific features of both host cell and replicon, whichfeatures limit the survival of the cell. In the present context, suchorganisms which are provided with specific genetic information in theform of a replicon carrying this information to exhibit specificphenotypical traits, are termed "primary host cells".

One conventional way of ensuring the biological containment of aspecific organism harbouring a recombinant DNA molecule is to limit itsability to propagate outside a defined environment. Typically, hostorganisms are used which have been attenuated by introducing a number ofindependent mutations resulting in well-defined requirements for one ormore growth factors which are not usually found in the naturalenvironment (defined as the environment outside the defined environmentof for instance a laboratory or production facility in the foliowingoccasionally termed the "outside environment"); the term "naturalenvironment" is intended to include the intestinal tract! and/or agenerally decreased competitiveness relative to wild-type organisms ofthe same species. For instance, E. coli K-12 is an attenuated bacterialstrain which is commonly used in experiments and productions involvinggenetic engineering as this attenuated strain is unable to propagate andestablish itself outside the defined conditions of the laboratory orproduction facility in which it is employed. Furthermore, this E. colistrain is unable to adhere to the epithelial cells of the mammalianintestinal tract which is the normal environment of E. coli which meansthat colonization of the natural habitat of E. coli by geneticallyengineered E. coli K-12 is highly unlikely to take place.

It should be noted, however, that even though E. coli K-12 is unable tocompete with natural organisms, it will still survive for a period oftime in a natural environment.

When the experiment or actual production involves the controlled releaseof a genetically engineered organism to the natural environment (asdefined above), it is not feasible to obtain biological containment byusing an attenuated host cell as described above. Obviously,microorganisms which are released to the environment in order tofunction there have to be able to compete favourably with the wild-typeorganisms in the same environment either of the same species or otherspecies in order to establish themselves, at least transiently, in asuitable ecological niche.

Another area of biological containment is concerned with limiting thespread of genetic information present on a replicon (optionallyincluding inserted foreign DNA), which replicon may for instance be abacterial plasmid, from a primary host cell used for experimentation orindustrial production to other cells of either the same species butlacking the attenuating mutations of the primary host cells imposed as apart of a biological containment system or to cells of a differentspecies which are able to propagate outside the defined environmentrequired for the growth of the primary host cells, which definedenvironment is part of a biological containment system.

Genetic information can be transmitted among organisms by several means.In the case of bacteria and bacterial plasmids, these may be transferredby bacterial conjugation, where a physical bridge is formed between twomating bacteria so that the plasmid passes from one bacterium to anothervia this bridge. Bacteria of different species may exchange plasmids byconjugation, and certain plasmids are in fact transmissible between suchdistantly related gram-negative bacteria as E. coli and Pseudomonas spp.As the ability of bacteria to conjugate and the ability of plasmids tobe transferred are properties which are associated with plasmid-borneDNA sequences, it is required that vectors to be used in industrialproduction involving genetically engineered bacteria lack the DNAsequences responsible for bacterial conjugation and plasmid transfer.This requirement constitutes the major biological containment measuretaken with respect to bacterial plasmids.

However, genetic information, which may for instance be present on abacterial plasmid, may also be spread by other means which are notcounteracted by the removal of said genetic information coding forbacterial conjugation and plasmid transfer.

Primary host cells (attenuated by the proper mutations to ensurelong-term survival under defined environmental conditions only)harbouring a recombinant DNA plasmid may occasionally be infected by oneor more naturally occurring bacteriophages. Some bacteriophages areknown to possess the ability to take up plasmids or other DNA moleculesat random and transmit them to secondary host cells (cells not intendedfor the production of biosynthetic products or other purposes, i.e.typically wild-type strains found in the natural environment) which havenot been attenuated and which are therefore capable of propagatingoutside the defined environment employed for growing the primary hostcells.

A similar situation may occur if a bacterium harbouring one of thenaturally occurring plasmids coding for bacterial conjugation andcapable of being transferred on conjugation, conjugates with a primaryhost cell already barbouting a recombinant plasmid. Homologousrecombination may then take place between the two plasmids resulting inthe transfer of the recombinant plasmid to another host cell.

A further way of spreading genetic information to cells which lack theattenuating mutations performed on primary host cells is the passiveuptake of free DNA by the cells, the so-called transformation. Manynaturally occurring microorganisms are able to take up free DNA. The DNAmay then be integrated into the chromosome of the novel, secondary hostcell or may replicate autonomously in the host cells which, due to theabsence of attenuating mutations, may multiply and establish themselvesoutside the defined environment of the laboratory or productionfacility. There is some evidence to suggest that substantial amounts ofbacterial plasmid DNA are released in biologically active form from, forinstance, E. coli cells during growth in a fermentor. This wouldindicate that the fermentation medium from which the cells have beenharvested presents a major source of plasmid DNA which may potentiallybe taken up by a secondary host cell by transformation, albeit at a lowfrequency, if the fermentation medium is released to the environment.The currently employed methods of biological containment do not proposeany solution to this problem.

In case of experiments or practical applications involving thecontrolled release of genetically engineered microorganisms to thenatural environment (as defined above), the spread of the replicon(optionally including inserted foreign nucleotide sequence(s)) byconjugation may be limited if the genes or nucleotide sequencesresponsible for conjugation are not located in the vector, cf. thediscussion above. However, this method of biological containment doesnot suggest any measures against the spread of, for instance, abacterial plasmid to novel, secondary host cells by transduction, byrecombination with transmissible plasmids or by transformation ofrecombinant DNA released from lysed recombinant organisms.

Although attempts have been made to design strains with increasedbiological containment properties, most if not all of these suffer fromthe disadvantage that they considerably affect the growth properties ofthe cells even under preferential conditions in the laboratory orproduction facility, and most often growth inhibition rather than cellkilling is obtained outside the defined environment.

The outline given above of the problems concerning the containment ofrecombinant organisms has mainly been concerned with bacteria; it shouldbe emphasized that similar arguments apply to eucaryotic organisms andviruses.

DISCLOSURE OF THE INVENTION

The present invention presents a novel approach to the concept ofbiological containment by making use of an active containment factor,namely a cell killing function which is expressed if primary host cellsharbouring a recombinant DNA molecule are subjected to novelenvironmental conditions or as a result of a random event, or if asecondary host cell receives the recombinant DNA molecule originallyharboured in the primary host cell. In some cases, the secondary hostcell is only killed under conditions inducing the expression of the cellkilling function.

Thus, the present invention relates to a replicon in which a nucleotidesequence encoding a cell killing function is regulatably expressed whenthe replicon is harboured in one type of host cell (primary host cell),so that cells harbourinS the replicon are killed under conditions underwhich the cell killing function is expressed, and the nucleotidesequence encoding the cell killing function is regulatably orconstitutively expressed when the replicon is harboured in another typeof host cell (secondary host cell), so that these cells harbouring thereplicon are invariably killed or killed under conditions under whichthe cell killing function is expressed.

In the present context, the term "replicon" denotes a segment of nucleicacid, e.g a bacterial plasmid, a bacterial chromosome, a procaryoticvirus, a eucaryotic plasmid, a eucaryotic virus, a eucaryoticchromosome, eucaryotic mitochondria or eucaryotic chloroplasts.

In the present context, the term "cell" is intended to indicate bacteriaand eucaryotic organisms such as unicellular organisms, e.g. yeasts orfungi, as well as multicellular organisms such as plants, animals orfungi, and cells derived from the tissues of multicellular eucaryoticorganisms such as plants, animals or fungi.

It should be noted that the replicon may be so designed that it is ableto bring about containment of primary host cells inside a definedenvironment as well as of the replicon itself. When the replicon isharboured in one type of host cell, namely the primary host cells, thenucleotide sequence encoding the cell killing function should beregulatably expressed; this implies that when the primary host cell issubjected to certain conditions, e.g. as present within a definedenvironment where its presence is desired either for reasons involvingthe production of a specific product or because it has other functionssuch as degradation of a pollutant, the nucleotide sequence encoding thecell killing function is not expressed, and the host cells remain viableand able to fulfil their function. However, when the primary host cellsare subjected to a specific change in environmental conditions, the cellkilling function is expressed to kill the primary host cells barboutingthe replicon.

It may also be possible, as part of the process of manufacturing aspecific product, deliberately to kill the primary host cells presentin, e.g., a fermentation vessel, by providing conditions under which thecell killing function is expressed. This procedure would be inaccordance with the requirements stipulated by certain healthauthorities that genetically engineered organisms must be killed beforeleaving the fermentation vessel.

The principle of the present invention of obtaining biologicalcontainment by introducing a replicon carrying a nucleotide sequenceencoding a cell killing function in a primary host cell may make itpossible to use a wild-type strain as the primary host cells, e.g. cellsused in the industrial production of a biosynthetic product. This hasthe important advantage over the use of mutated, attenuated strainswhich have hitherto been employed as a safety precaution as indicatedabove that it is not necessary to use specific growth conditions such asspecific media containing one or more particular growth factors requiredby the mutated organism for growth, thus reducing the cost of the mediaemployed and allowing a wider range of media components to be employed.Furthermore, the wild-type organisms may be better suited for geneticmanipulations or show improved fermentation properties, or they may beones which produce a specific, desired biosyntbetic product, but whichhave hitherto not been permitted for use in large-scale production.

Should the replicon become taken up by another type of host cell, thesecondary host cell, which is usually a wild-type organism found in thenatural environment to which the primary host cells or optionally amedium in which the primary host cells have been grown are released, thenucleotide sequence encoding the cell killing function may beregulatably or constitutively expressed; in either case, the secondaryhost cell will be killed when expression of the cell killing function isno longer repressed or inhibited.

In some cases, the size of the DNA fragment comprising the nucleotidesequence coding for the cell killing function is not significant for itsuse according to the invention. However, it is often preferred that thenucleotide sequence coding for the cell killing function is present on asmall DNA fragment which is advantageous in view of the fact that thecopy number of the replicon usually becomes lower when the total size ofthe replicon is increased. Accordingly, insertion of the DNA fragmentcoding for the cell killing function with the purpose of obtaining abiological containment does not lead to any substantial decrease in theyield of a desired biosynthetic product also encoded by the repliconwhen the DNA fragment encoding the cell killing function only comprisesa short sequence. Advantageous nucleotide sequences coding for a cellkilling function have a size of 1500 nucleotides or less, preferably1000 nucleotides or less, such as 500-200 nucleotides or less.

One way according to the invention in which the expression of the cellkilling function may be regulated is by providing a replicon in whichthe expression of the cell killing function is regulated at the level oftranscription. The regulation at the level of transcription may becarried out in various ways, but the regulation preferably takes placeby means of a promoter regulated by one or more factors. These factorsmay either be ones which by their presence ensure expression of thenucleotide sequence encoding the cell killing function or may,alternatively, be ones which suppress the expression of said nucleotidesequence sO that their absence causes the cell killing function to beexpressed. Thus, when a primary host cell is released to the surroundingenvironment or when a recombinant DNA molecule is taken up by asecondary host cell, i.e. outside the defined environment of experimentor production or a specific restricted environment to which an organismhas been released for a specific purpose, the promoter and optionallyits associated regulatory sequence is activated by the presence orabsence of one or more of these factors to effect transcription of thenucleotide sequence encoding the cell killing function whereby a cellkilling product is produced and the host cells are killed.

Factors regulating promoter activity may be selected from a wide varietyof factors. Principally, the expression of the gene encoding the cellkilling function may be determined by the environmental conditions orthe physiological state of the cells, or by a cyclical or stochasticevent. In the present context, the term "cyclical event" is understoodto mean a cyclically recurring event causing changes in certain factorsknown to be factors useful in influencing the expression of the cellkilling function such as temperature conditions, changes in lightintensity or hormonal changes. The term "physiological state of thecells" denotes factors such as cell density or growth phase of thecells.

Advantageous factors according to the invention, since these are mosteasily regulatable, are the presence or absence of a certain chemical inthe environment or the physical conditions in the environment such asthe temperature prevailing in the environment or other physicalcharacteristics (e.g. the intensity of the light in the environment).Thus, it is possible to envisage containment systems in which thenucleotide sequence coding for the cell killing function is expressedwhen a certain chemical present in the fermentation medium of theprimary host organism is not present in the environment to which theprimary host cell is released, i.e. when primary host cells areaccidentally released from, e.g., fermentation tanks to the surroundingenvironment, a factor required for the growth or survival of the cellsis no longer present, or the factor may be exhausted from the mediumwith the same effect. The promoter regulating the transcription of thenucleotide sequence coding for the cell killing function may also beactivated by a chemical which is not present in the fermentation mediumof the primary host organism, but which is present in the environment insufficient quantities to activate the promoter. Similarly, the promotermay be one which is activated by a shift in temperature, which, in thecontainment principle involving the replicons of the invention, usuallyimplies a shift from a higher temperature in a fermentation vessel orthe intestinal tract to a lower temperature prevailing in the outsideenvironment, or the intensity of light in that the promoter may be apromoter which is activated in the presence of light of a sufficientintensity, but inactive in the darkness prevailing in the fermentationvessel which is the defined environment of the primary host.

Where primary host organisms are ones which are released to the naturalenvironment in a controlled fashion, e.g. to a restricted area of landor to the intestinal tract of an animal, the regulatable promoter may beone which is regulated by chemical means, i.e. by the presence orabsence of a certain chemical in the environment of the cells, but ismost advantageously a promoter which is activated cyclically, e.g. bychanges in temperature, or by a stochastic event. The term "stochasticevent" is intended to indicate an event which occurs at random with acertain frequency per cell per generation or frequency per time unitwhich, according to the invention, results in the killing of the cellsin which the activation of expression of the killing function occurs.The stochastic event may be occasioned by periodic inversions of theregion carrying the promoter or excision of a sequence carrying anegative regulatory element. The effect of establishing cell killing bystochastic events is that the population of host cells will have adecreased competitiveness compared to populations of naturally occurringorganisms.

It should be noted that the promoter used to initiate transcription ofthe nucleotide sequence coding for the cell killing function ispreferably a promoter which is able to cause expression of thenucleotide sequence coding for the cell killing function in a wide rangeof host organisms in order to ensure a general applicability of theprinciple of the invention.

In case of a regulatable transcription of the cell killing function, theregulatory sequences may, for instance, be isolated from the bacterialoperons involved in the biosynthesis of amino acids or from bacterialgenes, the transcription of which is activated late in the stationarygrowth phase or from bacterial genes involved in the synthesis ofsurface structures (fimbriae). Examples of suitable promoters are E.coli trp which is activated in the absence of tryptophan, thebacteriophage λ P_(R) and P_(L) promoters controlled bytemperature-sensitive regulating factors, the B. subtilis sporulationgene promoters which are activated during sporulation, and the E. coliand Salmonella fimbriae gene promoters which are activatedstochastically.

In case of chemically regulatable promoters, the chemical, the presenceor absence of which determines the activation of the promoter, maysuitably be selected from carbon or nitrogen sources, metabolites, aminoacids, nucleosides, purine or pyrimidine bases or metal ions. When thechemical is one which, when present, suppresses promoter activity, itshould preferably be one which rarely occurs in the natural environmentin such concentrations that the promoter would not be activated when thehost organisms are released to the natural environment. One example of asuitable promoter in, e.g., an organism such as E. coli is the trppromoter which is repressed in the presence of a sufficientconcentration of tryptophan in the cell environment, but which isderepressed in the absence of sufficient quantities of tryptophan in theenvironment. A containment system using the trp promoter might thereforecomprise a quantity of tryptophan in, e.g., a fermentation vessel torepress the promoter which is derepressed when the host organisms arereleased from the fermentation vessel to the environment which usuallycontains very low concentrations or no tryptophan at all.

Promoters which are activated stochastically, by periodic inversions ofthe promoter region (in the present context, this is also termed an"invertible promoter" and "inversional switch promoter") and which areuseful for the purposes of the present.invention, also include the hin,cin and gin promoters (R.H.A. Plasterk et al., Proc. Natl. Acad. Sci.USA 80, 1983, pp. 5355-5358; G. Mertens et al., EMBO J. 3, 1984, pp.2415-2421; J. Zieg and M. I. Simon, Proc. Natl. Acad. Sci. USA 77, 1980,pp. 4196-4200). One invertible promoter which has been found to beparticularly useful due to its relatively small size is the fimApromoter which is one E. coli fimbriae gene promoter having thefollowing sequence: ##STR1## where the direction of transcription isfrom left to right and the proposed promoter consensus sequences areindicated at -35 and -10 (P. Klemm, EMBO J. 5, 1986, pp. 1389-1393).

The activation (inversional switch) of this promoter is regulated by thegene products of two genes which for the present purposes have beentermed the "on" gene and the "off" gene, the on gene product inducing aswitch from off (inactive) to on (active), and the off gene productinducing a switch from on to off. In a wild-type E. coli cell where thefimA gene and its associated promoter is present in one copy on thechromosome, the inversional switch occurs with a switching frequency ofone cell/1000 cells/generation. It is, however, possible to regulate thefrequency of the tnversfonal switch (substantially) according to need/asrequired by regulating the dosage of expression of the on and off genes.This may, for instance, be effected by means of suitable promotersinserted to transcribe into the on and off genes. The frequency oftranscription initiation by these promoters will then determine therelative dosage levels of the on and off genes formed. Thus, whenrelatively large amounts of the off gene product are formed, thefrequency of the inversional switch to the "on" position is lower thanwhen relatively larger amounts of the on gene product are formed.

An alternative way of obtaining host cell containment according to theinvention is to regulate the expression of the nucleotide sequencecoding for the cell killing function at the level of translation.

This may be done by providing an antisense RNA which inhibits thetranslation of the messenger RNA (mRNA) specifying the cell killingfunction in the primary host cell. The expression of the nucleotidesequence coding for the antisense RNA may be either constitutive orregulated, for instance to allow for an increase in the copy number ofthe replicon carrying the cell killing function, the only requirementbeing that the strength Of the promoter is such that sufficientquantities of antisense RNA are produced per unit time to completelyinhibit the translation in the primary host cell of the mRNA specifyingthe cell killing function. When such a replicon is transferred to anytype of secondary host cell in which the nucleotide sequence coding forthe cell killing function is transcribed and in which the product ofthat nucleotide sequence exerts a cell killing function, the absence inthe secondary host cell of the nucleotide sequence coding for theinhibitory antisense RNA results in translation of the mRNA specifyingthe cell killing function which in turn causes the death of thesecondary host cell. For all practical purposes, this means that theexpression of the nucleotide sequence coding for the cell killingfunction is regulated by the presence of the antisense RNA, the genesequence of which is suitably present on another replicon in the primaryhost cell.

In accordance with the invention, the expression of the antisense RNAmay be regulated as described above for the promoter initiatingtranscription of the nucleotide sequence coding for the cell killingfunction by a defined environmental factor influencing the activity ofthe promoter from which the nucleotide sequence coding for the antisenseRNA is transcribed. These environmental factors may be the same as thosementioned above, and comprise the presence or absence of a certainchemical in the environment, the temperature of the environment or theintensity of light in the environment of the primary host cell. Suitablepromoters may, for instance, be isolated from bacterial operons involvedin various catabolic pathways, in osmo-regulation or in heavy metalresistance. Suitable promoters activated by a chemical are the lac, araand deo promoters which are activated by the presence of lactose,arabinose and pyrimidine nucleosides, respectively, and osrA which isinduced in the presence of high concentrations of K⁺, and the promoterfor the mercury resistance gene of Tn501 which is induced by heavy metalions. When the antisense RNA is present in the primary host cell,translation of the mRNA specifying the cell killing function isinhibited through interaction between the two RNA species. However, ifthe primary host cell is released from its intended environment, theenvironmental conditions determining the promoter activity will bechanged so that the nucleotide sequence coding for the antisense RNAwhich has been designed to be expressed in a certain environment, willno longer be expressed, and the primary host cells will die. Similarly,if the recombinant DNA molecule carrying the nucleotide sequence codingfor the cell killing function is taken up by a secondary host mechanism,no antisense RNA will be present to prevent production of the cellkilling product, and the secondary host cells will also die.

If the nucleotide sequence encoding all or part of the antisense RNA isinserted between directly repeated nucleotide sequences of a sufficientsize, recombination between the repeats will occur in recombinationallyproficient cells with a frequency which to some extent can beexperimentally determined by varying the lengths of the repeats and/orthe distance between the repeats, leading to death of the cell whenrecombinational excision of the negatively acting regulatory elementtakes place. Apart from this, expression of the antisense RNA may alsobe regulated stochastically, for instance from an invertible promoter tobring about an inversional switch so that the antisense RNA is no longerexpressed. This promoter may advantageously be the E. coli fimApromoter.

Nucleotide sequences encoding a cell killing function to be inserted ina replicon of the invention may be derived from a wide variety ofsources such as bacterial plasmids, bacterial chromosomes, procaryoticviruses, eucaryotic plasmids, eucaryotic chromosomes, eucaryoticviruses, eucaryotic mitochondria or eucaryotic chloroplasts; they mayalso be produced synthetically according to standard procedures. Oneexample of a nucleotide sequence expressing a cell killing function isthe hok gene from the parB region of the plasmid R1, a region which haspreviously been shown to be involved in the stable maintenance of R1within a bacterial population, of. the disclosure of InternationalPatent Application No. PCT/DK83/00086, Publication No. W0 84/01172. Animportant feature of plasmid stabilization by parB has been found to bethe toxic effect of the hok gene product which is exerted if thetranslation of hok mRNA transcribed from the parB region of R1 is nolonger suppressed by a hok mRNA hybridizing antisense RNA, sok, which isalso transcribed from the parB region. Loss of the R1 piasmid from abacterial cell presumably leads to a change in the ratio between hokmRNA and sok RNA in the plasmid-free cell, presumably due to differencesin the half-life of the two RNA species, and ultimately to translationof hok mRNA when insufficient concentrations of the inhibitory sok RNAare present in the cell, which causes the death of the plasmid-freecell.

The nucleotide sequence coding for a cell killing function may becombined with promoter sequences such as those described above orcombined within the primary host cell with a sequence coding for anantisense RNA as described above. These sequences may be derived fromnatural sources such as those mentioned above for the cell killingfunction, or may be produced synthetically.

This natural system is utilized in accordance with the principles of thepresent invention to design a system of biological containment utilizingthe hok gene from R1 to confine recombinant organisms to a definedenvironment such as a fermentation vessel, to confine recombinant DNAmolecules or viruses to specific host cells or host cells in a definedenvironment and finally to confine, in time and space, environmentallyreleased recombinant organisms or vectors carrying recombinant geneticinformation.

In accordance with the present invention, host cell containment, such ascontainment of an E. coli host containing a recombinant DNA moleculesuch as a bacterial plasmid, may be obtained if the hok gene is insertedby standard recombinant DNA techniques together with DNA sequencescontaining a suitable promoter/regulatory region in such a way that thetranscription of the hok gene is, at least partially, controlled by thepromoter/regulatory sequences; if specific environmental conditionsdetermined by the nature of the promoter/regulatory sequences used arenot met, the promoter/regulatory region is derepressed, resulting intranscription of the hok gene which in turn leads to cell death.Alternatively, it may be possible to use another form of regulation, forinstance translational control as described above by using an antisenseRNA trnhibitton of hok mRNA translation. Such a system may be devised sothat the hok gene is constitutively expressed from the plasmid-bornegene while the translation of hok mRNA is counteracted by the synthesisof a properly designed antisense RNA, the gene coding for which isexpressed from a regulated promoter as described above whose activitydepends on the presence of one or more specific environmental factors.When these factors are no longer present, the promoter will no longer beactive, and therefore antisense RNA will no longer be expressed and nolonger inhibit the translation of hok mRNA so that the toxic product isformed and the host cells are killed.

As described above, the presence of the parB region (containing the hokand sok genes) on a plasmid stabilizes plasmid inheritance. This basicstabilization principle maybe utilized according to the presentinvention by inserting a regulatable, preferably strong promoterupstream of the hok and sok genes in such a way that transcription fromthe promoter results in synthesis of the Hok protein, because the hokmRNA is expressed in excess relative to the inhibitory sok antisenseRNA. Thus, under conditions where no transcription from the insertedpromoter takes place, the plasmids are stably maintained in the growingpopulation of cells, while under different conditions, e.g. in theoutside environment or in a secondary host cell, transcription of theinserted promoter takes place, and the cells are killed.

In accordance with the present invention, it is contemplated, in hostorganisms in which the R1 hok gene product will not be toxic, to ploysequences which are homologous to or related to the R1 hok gene fromother organisms which will be active in those organisms according to thesame principles as those established for the R1 hok gene product. Theterm "homology" is used here to denote the presence of any degree ofcomplementarity between a given probe and the nucleic acid species beinganalyzed. The "degree of homology" is expressed as the fraction ofcomplementary bases in a duplex nucleic acid molecule formed between agiven probe and the nucletc acid species being analyzed. The minimumdegree of homology which is detectable is a function of the experimentalconditions employed during hybridization and of characteristics of theprobe and the nucleic acid species being analyzed. Such homologoussequences have been found within the chromosomal DNA of a large numberof bacterial species (including gram-positive bacteria), within themitochondrial DNA of the yeast, Tetrahymena pyriformis and within humancells as well as within pea chloroplast DNA, all of which have a DNAsequence related to the R1 parB sequence as determined by DNA/DNAhybridization. Thus, the invention also relates to replicons which carrya nucleotide sequence which is homologous to the hok gene.

The present invention also relates to a primary host cell which harboursa replicon as described above. The cell may also comprise a nucleotidesequence coding for an antisense RNA inserted in the cell genome asdescribed above. The primary host cell maybe selected from a widevariety of cells such as bacteria or eucaryotic organisms such asunicellular organisms, e.g. yeasts or fungi, cells derived from thetissues of multicellular organisms such as plants, animals or fungi.

In a further aspect, the present invention relates to a nucleotidesequence which encodes a cell killing function. The nucleotide sequencemay further comprise a sequence regulating the transcription of asequence encoding the cell killing function. The regulatory sequence maybe a promoter with the features and the functions described above.

The invention further relates to a nucleotide sequence which encodes anantisense RNA capable of inhibiting the translation of an mRNAspecifying a cell killing function. As described above, this nucleotidesequence is preferably inserted into the cell on another replicon. Thenucleotide sequence coding for the antisense RNA may either be expressedconstitutively, or its transcription may be regulated from anothernucleotide sequence which, for instance, maybe a promoter regulated byone or more factors as described above.

In an important aspect, the present invention relates to a method ofcontaining a biological system, which comprises introducing into thebiological system a nucleotide sequence encoding a cell killing functionwhich sequence is regulatably expressed under certain conditions, andwhich is regulatably or constitutively expressed under differentconditions under which the biological system is maintained.

In the present context, the term "biological system" refers to anystructured biological material capable of reproduction such as nucleicacid (DNA or RNA) sequences, infectious agents such as viruses, bacteriaor unicellular eucaryotic organisms, e.g. yeasts or fungi, ormulticellular organisms such as plants, insects, etc., as well as cellsderived from the tissues of multicellular organisms. The term"containment" indicates that the spread of the biological system from aspecific restricted environment where specific conditions prevail andwhere its presence is desired, is limited or that the existence of thebiological system is limited to a certain period of time.

The containment is performed by maintaining the biological systems undercertain conditions which ensure that the cell killing function is notexpressed. These conditions may be intra- or extracellular, and maycomprise the phenotype and physiological state of the host organisms,host-vector relationships, the environmental conditions prevailing forthe biological system, or a cyclical event. When any one of theseconditions is changed, the cell killing function may be regulatably orconstitutively expressed so as to kill the host organism carrying thenucleotide sequence encoding the cell killing function. Additionally,the conditions comprise stochastic events.

When the biological system comprises cells, these may be contained underdefined environmental conditions by inserting into the cells anucleotide sequence containing a sequence encoding a cell killingfunction and a sequence regulating the transcription of the sequencecoding for the cell killing function, or, separately, a nucleotidesequence encoding a cell killing function and a nucleotide sequenceencoding an antisense RNA inhibiting, when expressed, the translation ofthe mRNA specifying the cell killing function, as described above.

In accordance with the principles of the present invention, thenucleotide sequence coding for the cell killing function is preferablycarried on a replicon. The nucleotide sequence coding for the antisenseRNA may be inserted in the cell on another replicon. The cells containedaccording to the method of the invention may be selected from bacteriaor eukaryotic organisms.

Apart from providing containment of host organisms to exist only underdefined conditions, the containment method according to the presentinvention also provides containment of a replicon to a primary host cellby inserting into the replicon a nucleotide sequence encoding a cellkilling function, the nucleotide sequence being regulatably transcribedfrom a regulatory sequence which is regulated by one or more factors, atleast one of which is encoded by a nucleotide sequence presentexclusively in the genome of the primary host cell.

Alternatively, the replicon may be contained to a primary host cell byinserting into a replicon a DNA fragment from which is constitutivelyexpressed an mRNA encoding a cell killing function, the translation ofwhich is inhibited by an antisense RNA transcribed from anothernucleotide sequence inserted into the primary host cell, the nucleotidesequence coding for the antisense RNA being constitutively expressed orexpressed from a promoter regulated by one or more factors such as oneof the factors described above. The replicon may also be so designedthat, apart from being contained to a primary host cell, it is alsocontained to cells of the same species and a definable range ofsecondary host cells, i.e. cells in which the factors responsible forregulating the expression of the cell killing function are also present.

As will be apparent from the above disclosure, the biologicalcontainment method of the present invention is a highly versatile methodwhich is applicable to a wide range of host cells and replicons to allowan active biological containment not only of attenuated organisms butalso of wild-type strains intended either for production of a specificbiosynthetic product or for release to the natural environment (theoutside environment or the intestinal tract of an animal); furthermore,by the present method, active containment of a given replicon to aspecific host is obtained.

It is further contemplated that the principle of the present inventioninvolving a replicon carrying a nucleotide sequence encoding a cellkilling function which is expressed under certain predefined conditions,may be utilized in the preparation of live vaccines. Such vaccines,based on non-pathogenic (e.g. attenuated) strains of otherwisepathogenic microorganisms or viruses have been known for a long time.Prominent examples of agents used in live vaccines are the vacciniavirus, the attenuated poliovirus (derived by Jonas Salk) and the BacilleCalmette-Guerin (attenuated Mycobacterium tuberculosis). Live vaccinesare advantageous in that they confer a prolonged, if not lifelong,immunit. y against the pathogenic agent in question. Furthermore, theyare generally cheaper and easier to administer than vaccines based oninactivated (killed) pathogens or purified proteins.

However, the use of live vaccines has been limited since it is oftendifficult to obtain the right combination of attenuation, viability andrelevant immune response. Furthermore, the deliberate release ofgenetically engineered bacteria to the environment, whether external orinternal, is currently not allowed in any country for reasons of publicconcern as to the possible long-term environmental impact, especiallythe risk of permanent establishment of the genetically engineeredbacteria in the environment.

The present invention has made it possible to circumvent the problemsassociated with the use of live vaccines by introducing in a suitablehost organism (a primary host cell as defined above) a nucleotidesequence encoding a cell killing function, the expression of which isdetermined by a stochastic event; a nucleotide sequence encoding adesired epitope for immunization (antigenic determinant) from apathogenic agent; as well as means for transporting the epitope, whenexpressed, to the outer surface of the cell, i.e. translocating itacross the cellular membrane systems. The nucleotide sequence encodingthe cell killing function and the nucleotide sequence encoding theepitope maybe present on the same replicon or on separate replicons. Inthis connection, the cell killing function may be any one of thoseindicated above. A currently preferred cell killing function is the oneencoded by the R1 hok gene.

The host cell may be any organism which is suited for being administeredto a mammal, e.g. a human being, to be immunized by the vaccine.Conveniently, the host cell is provided with genetic information for theexpression of adhesins, for instance a bacterium which, in nature,expresses adhesins by means of which they adhere to the surface ofepithelial tissue. (An adhesin may be defined as a structure responsiblefor adhesion of the bacteria to receptors present on epithelialsurfaces.) This is an important property of the host cell since itenables it to establish itself in a specific environment particularlyadvantageous for immunization purposes, e.g. where the type of theimmune response is optimal, i.e. secretory IgG and IgA, thus providing asuperior protection of epithelial surfaces. It should be noted that, inthis context, the term "environment" defined above as a specific,restricted environment where specific conditions prevail should beunderstood to include tissues and epithelial surfaces in the body aswell as cavities defined by such surfaces, such as the gastrointestinaltract, oral and nasal cavities, respiratory tract, urinary tract andreproductive organs. It is interesting to note that these areas coincidewith those first exposed to infectious (pathogenic) agents. It is atpresent contemplated that the vaccine may most conveniently beadministered as an oral vaccine, and consequently the host cell shouldin this case be one which is able to establish itself in the intestinesand compete successfully with the numerous organisms already present init.

Thus, examples of suitable primary host cells may be selected fromEnterobacteriaceae, e.g. E. coli, or lactic acid bacteria, e.g.Lactobacillus acidophilus, Vibrionaceae and Pseudomonades. The organism,however, need not necessarily be one which is inherently capable ofestablishing itself in the intestines. One may also select a hostorganism according to other criteria such as its suitability for beingsubjected to recombination techniques or fermentation procedures, andprovide it, by standard DNA recombination techniques, with genesexpressing adhesins, should the organism so selected lack such functionsenabling it to adhere to epithelial tissue.

The epitope for immunization may be introduced in the primary host cellsby inserting a nucleotide sequence encoding the epitope into a repliconin accordance with standard recombination techniques which are wellknown in the art (as described in e.g. Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982). Thus, thereplicon carrying the nucleotide sequence which codes for the epitopeshould further be provided with a suitable promoter, ribosomal bindingsite, translational initiation codon (ATG), to ensure expression of theepitope on the surface of the host cell. An essential feature of thevaccine according to the present invention is that the epitope should bepresented on the surface of the host cell in order to provoke anappropriate immune response in the mammal to be immunized. If theepitope is not naturally transported to the surface of the host cell,the nucleotide sequence encoding the epitope may be inserted into a genecoding for a naturally occurring cell surface protein, such as afimbrillin (the structural subunit of fimbriae), to express a fusionprotein which is translocated to the cell surface.

As indicated above, expression of the cell killing function is, in thecase of a vaccine according to the invention, determined by a stochasticevent which, as explained above, will typically be brought about by aperiodic inversional switch of a promoter to transcribe into thenucleotide sequence encoding the cell killing function, a promotersubjected to an inversional switch from inactive to active with afrequency which may, for instance, be regulated by the respective levelsof expression of an on and off gene, as explained above with referenceto the fimA promoter. It may be expected that similar promoters areregulated by similar mechanisms. This makes it possible to adjust thefrequency of the inversional switch so as to cause maintenance of asufficient dosage level of the epitope in question for the period oftime required to obtain a satisfactory immunization of the animal towhich the vaccine is administered. Alternatively, the stochastic eventmay be brought about by a periodic inversional switch of a promotertranscribing a nucleotide sequence encoding an antisense RNA inhibitingthe translation of the mRNA specifying the cell killing function, asexplained above. A currently preferred promoter is the E. coli fimApromoter. Apart from this, expression of the cell killing function maybe achieved by recombinational excision of the antisense RNA, asexplained above. The genes encoding the stochastic transcriptionmechanism (i.e. the promoter and optionally the on and off genes) of thecell killing function are conveniently inserted in the host cellchromosome rather than on a plasmid, for instance by means ofbacteriophages, in order to avoid loss of said genes as a result of lossof the plasmid from the cell. By regulating the frequency of theinversional switch, a certain predetermined percentage of the host cellswill be killed in each generation. This ensures that the cell populationcannot compete with the natural bacterial flora of, e.g., the intestinesover a longer period of time.

By allowing the organism to become established in the intestinalenvironment for such a predetermined period of time before the cellkilling function is expressed, it is possible to ensure that the dosageof the epitope to which the body to be immunized is exposed will besufficiently large and last for a sufficient period of time to providean adequate immunization. It is estimated that a satisfactoryimmunization may be obtained if the host cells are present in sufficientamounts in the defined environment for a period in the range of 15-30days, dependent on the nature and activity of the epitope expressed fromthe host cell.

In principle, the epitope expressed by the primary host cell may be anepitope from any pathogenic agent against which it is desired to obtainimmunity. Such pathogenic agents comprise viruses, bacteria oreukaryotic organisms such as fungi or protozoans. Examples of virusesfrom which epitopes to be used in connection with the live vaccine ofthe invention may be obtained, are viruses belonging to the familiesadenoviruses, herpetoviruses, papovaviruses, myxoviruses,orthomyxoviruses, paramyxoviruses, poxviruses, rhabdoviruses,arboviruses, or reoviruses. Other virus families of interest in thisconnection are the picornaviruses and retroviruses. Specific examples ofviruses are influenza virus, parainfluenza virus, measles virus, mumpsvirus, rubella virus, rhinovirus, rabies virus, HTLV I and II virus, HIVviruses, hepatitis B virus and other viruses causing hepatitis,poliovirus, rotavirus, reovirus, Epstein-Barr virus, Herpes simplex Iand II virus, cytomegalovirus, human papilloma viruses of various types,etc.

Examples of bacteria from which epitopes to be used in connection withthe live vaccine of the invention may be derived, are enteric bacteria,e.g. pathogenic strains of Escherichia coli, Salmonella spp. such as S.typhimurium, S. typhi, S. schottmulleri and S. choleraesuis, Vibriocholerae, Shigella dysenteriae; Corynebacterium diphteriae;Mycobacterium tuberculosis; Neisseria spp. such as N. gonorrhoeae, N.meningiditis and N. catarrhalis; Pseudomonas spp. such as P. aeruginosa;Yersinia spp. such as Y. pestis; Moraxella spp. such as M. bovis;Staphylococcus spp. such as S. aureus; Streptococcus spp. such as S.pneumoniae and S. pyogenes; Borderella spp. such as B. pertussis and B.bronchiseptica; Hemophilus influenzae; Treponema pallidum; andClostridium spp. such as C. botulinum and C. tetani.

Examples of pathogenic eukaryotic organisms, epitopes of which may beused in connection with the live vaccine of the invention, are fungi,e.g. Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioidesimmitis, Cryptococcus neoformans and Candida albicans; protozoans, e.g.Giardia lamblia; Trypanosoma spp. such as T. gambiense, T. rhodesienseand T. cruzi; Leishmania spp. such as L. donovani and L. tropica;Entamoeba histolytica; Naegleria spp.; Plasmodium spp. such as P.falciparum, P. vivax, P. malariae and P. ovale; and Isospora spp. suchas I. belli and I. huminis.

It is further contemplated that it will be possible to provide acombination vaccine against a variety of pathogenic agents byintroducing, in the host cell, two or more nucleotide sequences encodingepitopes from different pathogenic agents in such a way that theepitopes are expressed as fusion proteins together with a fimbrillin,substantially as described above or transported to the cell surface byother means, e.g. due to the presence of a signal peptide, to provide acombination vaccine. In this case, too, the epitopes will be exposed onthe surface of the host cell as parts of different fimbriae. Animportant advantage of this embodiment of the invention is thatimmunization may be effected simultaneously against a variety ofpathogens, only a single administration of the vaccine being required.

In order to avoid any risk of contaminating the outside environment(i.e. the environment outside the animal to be immunized with thevaccine of the invention) with live primary host cells which pass fromthe defined environment in the animal in question where their presenceis desired to the outside environment, for instance with the fasces incase of an oral vaccine, it should be possible to kill the host cellsonce they have passed into the outside environment. This may beaccomplished by inserting an additional promoter (apart from thestochastic promoter) into the host cell, which promoter, when activated,transcribes into a nucleotide sequence encoding a cell killing function,thereby causing death of the host cell or any other cell (secondary hostcell) which comes to harbour a replicon carrying the nucleotide sequencewhich codes for the cell killing function. In one embodiment, theadditional promoter, when activated, transcribes into the samenucleotide sequence encoding a cell killing function as the one theexpression of which is determined by a stochastic event. It would alsobe possible to insert this additional promoter to transcribe, whenactivated, into another nucleotide sequence encoding a second cellkilling function (which may be identical to the first one) inserted onthe same or another replicon as the nucleotide sequence coding for thefirst cell killing function. Activation of this additional promoteradvantageously occurs as a result of, for instance, a decrease intemperature to below body temperature (about 37° C.) or by chemicalinduction, as explained above.

In this way, the non-viability in the outside environment of geneticallyengineered bacteria used in the live vaccine of the invention isensured. In cases where the nucleotide sequence encoding the cellkilling function and the gene coding for the epitope are present on thesame replicon, the accidental spread of the recombinant replicon tosecondary host cells, in this case usually wild-type organisms found inthe outside environment, is substantially prevented. The presence of thenucleotide sequence coding for the cell killing function and the geneencoding the epitope on the same replicon therefore constitutes apreferred embodiment of the vaccine of the invention.

Apart from being useful in the preparation of live vaccines as describedabove, the principle of the present invention is further contemplated tobe applicable to the development of vaccines based on killed pathogens.Until now, such vaccines (in the following also termed "killedvaccines") have been known to be less efficient than live vaccines.Without wishing to be limited to any particular theory, the presentinventors believe that the diminished efficiency of killed vaccines maybe ascribable to the way in which the pathogenic agents used in thevaccines are inactivated, which is usually by heat treatment or chemicalinactivation with formaldehyde. This is thought to denature the antigenstructures of the pathogen in question, giving rise to a less adequateimmune response when the vaccine is administered and hence a lessthorough immunization.

This problem may be circumvented by utilizing the measures of thepresent invention. Accordingly, it may become possible to produce akilled vaccine which comprises a pathogenic agent carrying one or morenucleotide sequences encoding a cell killing function, which pathogenicagent has been killed by the expression of one or more of saidnucleotide sequences. In this way, the antigen structures of thepathogen will remain intact so that, theoretically, a more efficientimmunization of the mammal to which the killed vaccine is administeredwill be obtained. The patbogen employed in the killed vaccine may be anyone (or a combination) of those listed above as providing the genescoding for epttopes to be introduced in live non-pathogenic host cellsfor use as live vaccines.

Because of a low but definite risk of mutations affecting the killingfunction, this type of vaccine may only be of practical relevance in thefield of veterinary medicine.

The expression of the cell killing function may be regulated at thelevel of transcription, e.g. by means of a regulatable promoter. Any oneof the promoters discussed above may be employed. Alternatively, theexpression of the cell killing function may be regulated at the level oftranslation as discussed above, e.g. by means of an antisense RNAinhibiting the translation of the mRNA specifying the cell killingfunction.

In a specific embodiment of the killed vaccine of the invention, thevaccine, when administered, comprises live pathogenic agents into whichhas been inserted a cell killing function which is activated in the bodyas a result of the environmental changes to which the pathogens aresubjected, e.g. changes in temperature, pH or the presence of certainchemicals.

The vaccines of the invention (live or killed) may be formulated fororal or parenteral administration in accordance with usual practice inthe field of human and veterinary medicine together with apharmaceutically or veterinarily acceptable carrier or vehicle.

For oral administration of a live vaccine, it is preferred to protectthe host cells against the gastric environment which tends to bedetrimental to the viability of, e.g., many bacteria contemplated to beuseful for the present purpose. This protection may, for instance, beprovided in the form of an enteric coating.

1. Gramnegative and grampositive bacteria

Suitable replicons for genetic engineering in bacterial host cells mayfor example be plasmids capable of replicating in Enterobacteriaceae,e.g. pBR322 or R1 runaway replication plasmids (European PatentApplication No. 83305438.0, Publication No. 0 109 150), or capable ofreplicating in gramnegative.bacteria in general, e.g. plasmids derivedfrom RSF1010 (Bagdasarian et al., Gene 16, 1981, pp. 237-242), orplasmids capable of replicating in grampositive bacteria such as B.subtilis, e.g pC194 and pUB110 (Lovett and Keggins, Meth. in Enzymol.68, 1979, pp. 342-357). In order to biologically contain such bacterialplasmids or cells containing such plasmids according to the invention,the DNA fragment or DNA fragments comprising the R1 hok region can beinserted into the replicon in such a way that the R1 hok expression isgoverned by regulatable promoter(s) known to be recognized in the hostcell in question, such promoters being either natural promoters orsynthetic promoters, such as the E. coli trp promoter or the B. subtilispromoters governing expression of certain genes in stationary phasecells. As shown in the Examples, the R1 hok gene product is toxic in awide range of gramnegative bacteria as well as in B. subtilis (cf.Example 16) and hence probably in all grampositive bacteria. If the R1hok gene product is not lethal to the host cell in question--a definiterequirement in order to establish the biological containment system--anR1 hok homologous sequence can be isolated from either the genome of thehost cell in question (or a closely related bacterial species), from aplasmid naturally occurring in the host cell in question (or a closelyrelated bacterial species), or from a bacterial virus and subsequentlytested for hok-like activity in a manner similar to that described inthe Examples for one E. coli chromosomal homologue of R1 hok.

Establishment of a biological containment system for, e.g., fermentationpurposes involving the use of R1 hok or a nucleotide sequence homologousto the hok in bacteria thus includes: selection of replicon and hostcell; insertion into the replicon of the proper sequence comprising R1hok or a nucleotide sequence homologous to the hok which is notexpressed in the selected host cell under defined conditions; insertioninto the replicon of a gene or genes encoding the useful product(s) tobe produced in large quantities; introduction of the recombinantreplicon into the bacterial host cell by standard techniques ofbacterial transformation; cultivation of the replicon-containing hostcells in a culture medium supplemented with the necessary nutrientsincluding any exogeneous factor required for the containment system inquestion for the number of generations required to reach the desiredcell concentration; and finally, harvesting of the cells and the mediumfrom either of which the product in question can be isolated. If thecells are accidentally released to the outside environment, the promoterregulating the transcription of the hok or hok-like sequence will beactivated, and the cells will be killed as a result of the expression ofthe hok or hok-like product or the promoter regulating the transcriptionof the antisense RNA is inactivated. Similarly, if DNA from the cells istransferred to other cells (secondary hosts), the promoter regulatingthe transcription of the hok or hok-like sequence is activated, and thecells are killed, which is also the case when the hok or hok-likesequence is regulated by an antisense RNA, in cells lacking a nucleotidesequence coding for the antisense RNA.

2. Yeast cells

The technical exploitation of recombinant DNA techniques in eucaryoticsystems may be desired to obtain such post-translational modifications(specific proteolytic cleavages, glycosylation, etc.) of primary(eucaryotic) gene products that are not carried out in bacteria or are,at best carried out in a suboptimal manner. A widely used eucaryoticorganism is the yeast Saccharomyces cerevisiae in which a naturallyoccurring plasmid, the 2 μ replicon, has been adapted as a vector forexpression of genes not naturally related to the 2 μ replicon in S.cerevisiae. As described above, it is possible to isolate or construct asequence to be inserted into a yeast replicon, e.g. the 2 μ replicon,utilizing the principle of the R1 hok biological containment mechanismfor containing yeast cells and plasmids.

Although the native promoters of R1 hok are not likely to be utilized inS. cerevisiae cells, the conservation of hok-like sequences in organismswhich are only distantly related and the toxicity of R1Hok togrampositive as well as gramnegative bacteria makes it reasonable toassume that the product of the R1 hok gene and of genes related to R1hok (e.g. relB-orf3 or par1 or other genes originating from bacterialgenomes which show a homology at the sequence and functional level to R1hok or similar genes isolated from bacterial plasmids) should be testedfor their ability to kill yeast cells, such as S. cerevisiae. Inpractice, this will entail isolating the coding region of the hok geneor hok-like gene and linking the coding region to a suitable regulatableyeast cell promoter, the resulting replicon being finally introduced, bystandard methods, into yeast cells, and the effect of expression of thehok or hok-like gene is investigated. If cell death ensues, a usable hokor hok-like gene has been identified.

Alternatively, sequences identified in DNA from yeast cells withhomology to parB or relB-orf3 can be isolated, linked to a proper yeastcell promoter, inserted into the 2 μ replicon and following introductionof the recombinant replicon into S. cerevisiae, tested for their abilityto kill the cell. From a hok gene or a hok-like gene shown to be toxicupon expression for e.g. S. cerevisiae, a biological containment systemidentical to or analogous with the R1 hok system can be generated byimposing a regulatory loop (a regulatable promoter or a gene encoding anantisense RNA regulated by a proper yeast promoter) as previouslydiscussed in the description of the general strategy. The resultingregulatable yeast hok sequence or hok-like sequence can be inserted inany yeast replicon, e.g. the 2 μ replicon or derivatives thereof intowhich genes not naturally related to 2 μ have been inserted with theintention of obtaining expression of the inserted genes, with thepurpose of biologically containing cells and/or recombinant replicons.The replicon can be introduced into yeast cells, e.g. S. cerevisiaecells, by transformation or protoplast fusion, and following selectionof cells carrying the replicon, these can be further grown into alarge-scale culture in the appropriate culture medium supplemented withthe necessary nutrients as well as any exogeneous factor(s) required forthe containment system in question. The culture of cells harbouring thereplicon in question is then harvested, and any useful product expresedfrom the replicon can be isolated from either the yeast cells or theculture medium, depending on the gene and the gene product in question.If the cells are accidentally released to the outside environment, thepromoter regulating the transcription of the hok or hok-like sequencewill be activated, and the cells will be killed as a result of theexpression of the hok or hok-like product or the promoter regulating thetranscription of the antisense RNA is inactivated. Similarly, if DNAfrom the cells is transferred to other cells (secondary hosts), thepromoter regulating the transcription of the hok or hok-like sequence isactivated, and the cells are killed, which is also the case when the hokor hok-like sequence is regulated by an antisense RNA, in cells lackinga nucleotide sequence coding for the antlsense RNA.

3. Mammalian cells

The requirement for specific post-translational modifications maynecessitate the expression of certain eucaryotic genes in mammaliancells, i.e. of human or animal origin, rather than In bacteria or yeastcells. Replicons that can be used as cloning vectors in eucaryotic cellsare derived from chromosomes (ars replicons) from DNA viruses, e.g. SV40and bovine paptlloma virus, or from RNA viruses, e.g. retroviruses. Thetwo DNA viruses mentioned can be maintained in a plasmid state ininfected cells while most retroviruses (RNA-containing viruses) need tobe genetically modified in order to exist as freely replicating DNAmolecules rather than as chromosomally integrated copies of the viralDNA genome. It may be anticipated that large-scale cultures of cellscontaining the above-mentioned replicons into which a gene or genes notnaturally related to the replicon has been inserted with the aim ofobtaining expression of a useful product, will need to be contained asdiscussed in the section on procaryotic vectors.

In a manner similar to that described under the yeast cell system, asequence containing a regulatably expresed R1 hok gene or a nucleotidesequence homologous to hok can be constructed, once a gene has beenidentified that exerts a hok or a hok-like effect in the host cell inquestion. A first step would thus be to insert the coding sequence ofknown hok or hok-like genes, irrespective of their origin, frombacterial plasmids, bacterial genomes or yeast cell genomes, into areplicon capable of replicating in the host cell in question in such away that expression of the hok gene is obtained upon induction ofexpression, i.e. supplemented with all necessary regulatory sequences asis required for expression of a gene in the host cell in question. Apromoter sequence suitable for insertion upstream of the hok or hok-likegene would be the mouse mammary tumor virus LTR (long terminal repeatsequence) which is inducible with steroid hormones or the regioncontrolling the expression of the metallothionein gene which isinducible with metal ions. If cell death ensues upon induction oftranscription, a hok gene or a hok-like gene has been identified for thehost cell in question, and from this hok or hok-like gene, a repliconbiological containment system can be constructed by a regulatory loop atthe transcriptional/translational level as described above.

If none of the available hok or hok-like genes of bacterial or yeastorigin exert a toxic effect in the mammalian host cells in question,novel hok-like sequences maybe isolated from a mammalian genome (e.g.the sequences discovered in Tetrahymena mitochondrial DNA and in humancellular DNA) and subsequently tested for hok-like activity whenproperly expressed. The recommended strategy for the detection of novelhok-like sequences has been outlined above.

The use of the hok-like containment mechanism in mammalian cells willthus include: selection of replicon, e.g. a retrovital vector andselection of host cells which will depend upon the actual sequencesgoverning the expression of the inserted hok-like nucleotide sequences;insertion into the replicon of such foreign genes which code for theuseful product(s) to be produced into the replicon; introduction of therecombinant replicon into the mammalian cell type in question bystandard techniques of DNA transfection or micro-injection; selection ofcells containing the replicon in question; growth of the cells in aculture medium adapted for the cell type in question by the addition ofnecessary nutrients and growth factors as well as any exogeneous factorrequired for the containment system in question with the intention ofobtaining a large-scale culture of cells expressing the gene encodingthe useful product; and, finally, the culture can be harvested and theuseful product isolated. If the cells are accidentally released to theoutside environment, the promoter regulating the transcription of thehok or hok-like sequence will be activated, and the cells will be killedas a result of the expression of the hok or hok-like product or thepromoter regulating the transcription of the antisense RNA isinactivated. Similarly, if DNA from the cells is transferred to othercells (secondary hosts), the promoter regulating the transcription ofthe hok or hok-like sequence is activated, and the cells are killed,which is also the case when the hok or hok-like sequence is regulated byan antisense RNA, in cells lacking a nucleotide sequence coding for theantisense RNA.

While particular types of replicons adapted for particular types ofcells have been discussed in the detailed sections above, the generalprinciple of utilizing the containment mechanism of the invention is thesame, irrespective of the type of replicon and cell barbouring thereplicon: the establishment of a host killing function and a regulatoryfunction adapted to regulate the expression of the cell killing functionin cells harbouring the replicon so that, under conditions where thecell killing function is expressed, the host cell is killed.

DESCRIPTION OF THE DRAWINGS

Reference is now made to the drawings, where

FIG. 1 shows a deletion mapping of the parB⁺ region. The localization ofthe parA⁺ region and the parB⁺ region within the EcoRI-A fragment ofplasmid R1 are shown as black boxes. Restriction enzyme sites in theEcoRI-A fragment are as described in International Patent ApplicationNo. PCT/DK83/00086, Publication No. WO 84/01172. The parB⁺ region islocated within the 1.9 kb PstI fragment bordered by coordinates 15.0 to16.9. The parB⁺ region was further mapped to the right-hand 580 bp of an880 bp RsaI fragment. The cross-hatched region indicates the minimalparB⁺ region. The position of the hok and sok genes within the 580 bpparB⁺ region is also shown. A BglII-SalI fragment containing the λpRpromoter and the cI857 allele of the λ repressor gene was inserted intopBR322 derivatives carrying various parts of the parB fragment. Theposition of the inserted fragments and the direction of transcriptionfrom λpR are shown below the map of the parB⁺ region (arrows). The λpRpromoters in pKG633, pKG634 and pKG341 read from left to right into theparB⁺ region whereas the λpR promoter in pKG171 reads from right toleft. Restriction enzyme sites are shown as E (EcoRI), B (BalI), B₂(BglII), S (SalI), R (RsaI), and P (PstI).

FIG. 2 shows a map of plasmid pPR95 (13 kb). copA, copB representsreplication control genes of plasmid R1; repA represents a gene requiredfor R1 replication; ori is the origin of replication; bla denotes a geneconferring ampicillin resistance on plasmid-carrying cells; parBrepresents the R1 derived maintenance function encoding the hok and sokgenes; deo-lacZ denotes a translational fusion between the deoC gene andthe IacZ gene. lacZ,Y,A represent the lac operon; cI857 represents agene which codes for a temperature sensitive λ repressor controlling λpRpromoter activity. Arrows denote direction of transcription. The blackbars denote the extension of the various genes. Restriction enzyme sitesare shown as SalI (S), BglII (B₂), BamHI (B) and EcoRI (E).

FIG. 3a and 3b shows the nucleotide sequence of the parB⁺ region. The 5'end of the upper DNA strand is positioned at right. The numbering of thebases are in accordance with the coordinates of the parB⁺ region inFIG. 1. Ter denotes the stop codons of the only three open readingframes present in the nucleotide sequence consisting of more than 50codons. fMet corresponds to the start codons of the same three openreading frames. The amino acid sequence of the hok gene product,starting at position +304, is shown below the DNA sequence--amino acidabbreviations are standard nomenclature. The underlined sequencesdesignated "-10" and "-35" is the promoter structure for the sok gene.

FIG. 4. shows host cell killing after λpR induced activation of the hokgene. Strain JC411 containing either pKG634 (closed symbols) or pKG171(open symbols) was grown exponentially in A+B minimal mediumsupplemented with casamino acids at 30° C. At time zero, the temperaturewas shifted to 42° C. and growth of the cultures was followed as OD₄₅₀and viable counts on selective medium (LB plates containing 50 μg/mlampicillin).

FIG. 5 is a photograph of cells sampled 1 hour after shift of strainJC411 (pKG634) to 42° C. Arrows point at cells with clearly changedmorphology. Cells with a normal morphology are also seen.Magnification×2000.

FIG. 6 shows the suppression of host cell killing. Strain JC411containing either pF634 alone (closed symbols) or pF634 plus pPR633(open symbols) was grown exponentially in A+B minimal mediumsupplemented with casamino acids at 30° C. At time zero, the temperaturewas shifted to 42° C. and growth of the cultures was followed bymeasuring the optical density (OD₄₅₀) and viable counts on selectivemedium (LB plates containing 100 μg/ml kanamycin).

FIG. 7a is a comparison of the amino acid sequences of the hok geneproduct and the relB-orf3 gene product. Conserved amino acids are withbold face types; amino acids representing conservative changes areunderlined.

FIG. 7b shows the alignment of the nucleotide sequences of parB and orf3of the E. coli relB operon (Bech et al., The EMBO Journal 4 , 1985, pp.1059-1066). The parB sequence is the upper strand, relB-orf 3 the lower,coordinates as in FIG. 3. Vertical bars indicate conserved nucleotides.Numbers in brackets are coordinates of the relB nucleotide sequence asgiven by Bech et al. The two sequences are aligned so that the startcodons of the two reading frames are at the same position--this isindicated with Met at position +304. The termination codons of the tworeading frees are indicated with Ter at position +460.

FIG. 8a shows 0.75 μg of EcoRI-restricted total DNA from strains of E.coli analyzed by filter hybridization using the R1 parB probe. Lane 1:R1drd-19; lane 2: R100; lane 3: R386. These lanes were exposed for 30minutes. Lane 4: RP1; lane 5: R6-K; lane 6: plasmid-free E. coli. Theselanes were exposed for 5 hours. Sizes of relevant fragments are given inkilobases.

FIG. 8b shows 0.75 μg of EcoRI-restricted total DNA from strains of E.coli analyzed by filter hybridization using the relB-orf3 probe. Lane 1:R100; lane 2: R386; lane 3: plasmid-free E. coli. Time of exposure: 3.5hours. Sizes of relevant fragments are given in kilobases.

FIG. 9 shows 0.5-0.75 μg of EcoRI-restricted total DNA from variousbacteria analyzed by filter hybridization using the R1 parB probe. Theautoradiogram was exposed for 17 hours. Two different photographicexposures of the same autoradiogram are shown: Lane 1: Salmonellatyphimurium (not discussed in the text); lane 2: Serratia marcescens;lane 3: Pseudomonas fluorescens; lane 4: Pseudomonas putida; lane 5:Proteus vulgaris (not discussed in the text); lane 6: Escherichia coli;lane 7: Bacillus subtilis; lane 8: Bacillus circulans PL236. Sizes ofradioactively labelled marker (λ restricted with HindIII) are given inkilobases.

FIG. 10 shows 0.5-0.75 μg of EcoRI-restricted total DNA from variousbacteria analyzed by filter hybridization using the relB-orf3 probe. Theautoradiogram was exposed for 17 hours (lane 19) and 72 hours (lanes2-7). Lane 1: Serratia marcescens; lane 2: Pseudomonas fluorescens; lane3: Pseudomonas putida; lane 4: Bacillus subtilis; lane 5: Bacilluscirculans PL236; lanes 6, 7: Lactobacillus. Sizes of radio-activelylabelled marker (λ restricted with HindIII) are given in kilobases.

FIG. 11 shows filter hybridization analyses of DNA from eucaryotic cellsusing the relB-orf3 probe (lanes 1-4) as well as the R1 parB probe(lanes 5-6). The DNA was cleaved with EcoRI (lanes 1-3 and 5-6) or withPstI (lane 4). Lane 1: 5 μg of macronuclear DNA from Tetrahymenathermophila; lane 2: 5 μg of total DNA from Tetrahymena thermophila;lane 3: 0.25 μg of chloroplast DNA from Pisum sativum; lane 5: 5 μg oftotal cellular DNA from neuroblastoma; lane 6: 10 μg of total cellularDNA from embryonic liver. Sizes of fragments are given in kilobases.

FIG. 12 shows a partial map of plasmid p341-1. The region presented hereis the fusion between the hok gene and the promoter region from the trpoperon of E. coli K-12 (obtained from plasmid pSGS8). In addition to thetrp promoter (indicated by the arrow), the NH₂ terminal end of the trpEgene is also present (indicated as trpE). The broken lines representpBR322 sequences from which the Ap^(R) gene and the origin ofreplication are indicated. Restriction enzyme sites are shown as E₁(EcoRI), B₁ -E₅ (fusion of BamHI (filled in by DNA polymerase I) withEcoRV) and X-S (fusion of XhoI and SalI).

FIG. 13 shows growth curves for MC1000 (p341-1) (circles) and MC1000(triangles) grown in A+B minimal medium supplemented with 0.2% glucoseand 1% casamino acids at 37° C. The cell density is measuredspectrophotometrically as OD₄₅₀.

FIG. 14 is a graph showing viable counts (OD₆₀₀) of E. coli HB101harbouring pNL7 (circles) or pBR322 (triangles), as a function of time.No exogenous tryptophan was added to the MA+B culture medium.

FIG. 15 is a graph showing viable counts (OD₆₀₀) of E. coli MB101harbouring pNL7 (circles) and pBR322 (squares), as a function of time. 5μg/ml tryptophan was added to the MA+B culture medium.

FIG. 16a shows a deletion map of the minimal parB⁺ region. The numberingis in accordance with the coordinates of the parB⁺ region shown inFIG. 1. The hok and sok genes within the region are indicated withfilled-in and open areas, respectively. The presume sok promoter isindicated as ← and the putative hok Shine-Dalgarno sequence is shown as*. The plasmids pPR341 and pPR345 are pBR322 derivatives, which containthe parB region from +268 to +580 and +303 to +580, respectively. Theplasmid pPR341 carries the hok Shine-Dalgarno sequence and the hokreading frame, whereas pPR345 only carries the hok reading frame. Bothplasmids are devoid of the sok gene. Restriction enzyme sites are shownas B₁ (BamHI) and E (EcoRI).

FIG. 16b shows the physical and genetic map of the plasmid pKG345 usedfor the induction of the cro -hok gene fusion. Plasmid pKG345 is apPR345 derivative in which a BglII-SalI fragment containing the λpRpromoter and the cI857 allele of the λ repressor gene was inserted intopPR345 restricted with BamHI and SalI. This construction placed thetranscription and translation of the hok gene under the control of theλpR promoter and cro Shine-Dalgarno sequence, respectively. The genefusion is indicated as an open area for the cro gene and as across-hatched area for hok. The bla gene, the λ repressor (filled-inareas) and the origin of replication are also indicated. The λpRpromoter is shown as ← and the cro Shine-Dalgarno sequence as *.Restriction enzyme sites are shown as R (RsaI), S (SalI), E (EcoRI), B(BamHI) and B₂ (BglII).

FIG. 17 shows host cell killing after λpR induced expression of the hokgene and the cro-hok⁺ gene fusion. E. coli strain MC1000 was grownexponentially in A+B minimal medium supplemented with 0.2% glucose and1% casamino acids at 30° C. containing either pKG341 (open symbols) orpKG345 (filled-in symbols). At time zero, the temperature was shifted to42° C. and growth of the cultures was followed as OD₄₅₀ and viablecounts on selective medium (LB plates containing 100 μg/ml ampicillin).

FIG. 18 is a map of plasmid pLK26. The filled-in areas denote structuralgenes; the insert shows the spacI promoter followed by a syntheticribosomal binding site and a polylinker; ori denotes the origin ofreplication from pBR322 and pUB110, respectively; lac o denotes the lacoperator.

FIG. 19 is a graph showing viable counts (OD₆₀₀) of B. subtilis BD170containing pSI-1 (circles) or pLK26 (squares), as a function of time.The cells were grown exponentially in LB medium with 5 μg/mlchloramphenicol at 37° C.

FIG. 20 is a graph showing the killing kinetics after induction of hokwith 2 mM IPTG. B. subtilis BD170 containing pSI-1 (circles) or pLK26(squares) were grown in LB medium with 5 μg/ml chloramphenicol. Viablecounts were monitored on LB plates with 5 μg/ml chloramphenicol.

FIG. 21 shows a map of plasmid pPKL8 (5.5 kb). The position of the fimB,fimE and the truncated fimA gene is indicated. The box with doublearrows denotes the invertible 300 bp region containing the promoter ofthe fimA gene. The hatched area indicates pBR322 DNA.

FIG. 22 shows a map of plasmid pPR341 (4.3 kb). The hatched areaindicates pBR322 DNA.

FIG. 23 shows a map of plasmid pPKL100 (7.5 kb). See FIGS. 14 and 15 fordetails.

FIGS. 24a and 24b show microphotographs of E. coli K-12 strain MC1000cells harbouring plasmid pPKL100. The arrows indicate killed ghostcells.

FIG. 25 shows digestions with SacII and SnaBI of plasmids pPKL100 (laneA) and pPKL8 (lane B). Lane C is a HindIII digest of bacteriophagelambda used as a molecular weight marker showing the following sizes:23.1 kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3 kb, 2.0 kb and 0.56 kb. The arrowsindicate fragments affected by the inversion of the 300 bp segment.

FIG. 26 shows maps of plasmids pLP4 (=A), pLP5 (=B) and pLP6 (=C). Thehatched boxes represent pACYC184 DNA. Relevant restriction sites as wellas the positions of the fimB and fimE genes are shown.

MATERIALS AND METHODS Bacterial Strains and Plasmids

The bacteria and plasmids are listed in Table 1.

The experimental techniques used were standard techniques employed inthe fields of microbial genetics (J. Miller: Experiments in MolecularGenetics, Cold Spring Harbor, N.Y., 1972) and genetic manipulation(Davis, Bothstein and Roth: A Manual for Genetic Engineering; AdvancedBacterial Genetics, Cold Spring Harbor, N.Y., 1980, and Maniatis,Fritsch and Sambrook: Molecular Cloning, Cold Spring Harbor, N.Y., 1982.

All cells were grown in LB medium (Bertani, J. Bact 62, 1951, p. 293)with 0.2% of glucose and 1 μg/ml of thiamin, or A+B minimal medium(Clark and Maal.o slashed.e, J. Mol Biol. 23, 1967, p. 99) supplementedwith 0.2% of glucose and 1% casamino acids. The plates used were LAplates containing LB medium and 1.5% of agar.

Clear lysates were prepared according to the method described by Clewelland Helinski, Proc. Natl. Acad. Sci. USA 62, 1969, pp. 1159-66.

Small scale preparation of plasmid DNA was performed by the method ofBirnboim et al., Nucl. Acids Res. 7, 1979, pp. 1513-23.

Large-scale preparation and analysis of plasmid DNA was performed usingdye boyant density gradient centrifugation according to Stougaard andMolin, Anal. Biochem. 118, 1981, p. 181.

The restriction endonucleases were used in accordance with theprescriptions provided by the manufacturer (Boehringer, Mannheim orBiolabs, New England) at 37° C. Double and triple digests were performedby starting with the enzyme requiring the lowest salt concentration andthen adjusting with additional buffer before adding the next enzyme.

Treatment with the exonuclease Bal31 was performed as follows: 0.1 unitof Bal31 was added to 50 μg linear DNA and samples were taken out at 1',2', 4', 8', 16', 32' and 60' to 60 mM EDTA, extracted with phenol,ethanol precipitated and resuspended in 20 μl TE buffer. Half of the 20μl was digested with the appropriate restriction enzyme subjected toagarose gel electrophoresis to determine the average size of the deletedDNA deletions. To the other half, the appropriate linker was added andthe mixture ligated for 48 hours with an excess of T4 DNA ligase.

Ligation of restricted plasmid DNA was performed as recommended by themanufacturer with the exception of blunt end ligation, where an excessof T4 DNA ligase and ATP was added.

pKG633: The SalI-BglII fragment of pOU82 containing the cI857temperature sensitive allele of the λ repressor gene and the λpRpromoter was inserted into pPR633 in front of the parB⁺ region so thatthe λpR promoter reads into the region from left to right (FIG. 1). Inan analogous way, the SalI-BglII fragment of pOU82 was inserted intopPR634 and pPR341, which are Bal31 deletion derivatives of pPR633,resulting in pKG634 and pKG341. pKG171: In pPR171, the SalI-BglIIfragment of pOU82 was inserted in the opposite orientation, resulting inpKG171. The positions and orientations of the inserted λpR promotersrelative to the hok and sok genes are shown in FIG. 1. pF634: TheEcoRI-SalI fragment of pKG634 containing the right 390 bp of the parB⁺region and the λ cI857-pR inducible promoter system was inserted intothe unique SalI site in the kanamycin resistance (aphA⁺) fragment ofpML31 by blunt end ligation (S1 nuclease was used to make the restrictedDNA fragments blunt-ended).

The DNA was cleaved with the appropriate restriction endonucleasesaccording to the recommendations given by the manufacturers. Forcellular DNA, 10 units per microgram of DNA was used. The incubationtime was 3 hours at 37° C. The generated DNA fragments were separated byelectrophoresis through 0.7% or 1% agarose gels in a Tris-acetate bufferat 0.8 volt per cm for 18 hours and visualized by ethidium bromidestaining.

Mobilization of Plasmids

E. coli S 17.1 is capable of mobilizing plasmids like RSF1010 due to aninserted conjugative plasmid (RP1 derivative) in the chromosome. Theplasmids in question were transformed to S 17.1 which then representedthe donors.

One drop of donor cells and recipient cells were mixed on an LB plate(no selection) and incubated overnight. From the resulting cell mass, aliquid suspension was made from which dilutions were spread ondouble-selection plates.

                  TABLE 1                                                         ______________________________________                                        Bacteria and plasmids                                                         Relevant phenotype                                                            ______________________________________                                        Bacterium                                                                     E. coli K-12, MC1000.sup.1)                                                                 Leu.sup.-, Lac.sup.-, Str.sup.R                                 E. coli K-12, S 17.1.sup.2)                                                                 Pro.sup.-, Str.sup.R, Mob.sup.+                                 E. coli K-12, 1005.sup.3)                                                                   Met.sup.-, Na1.sup.R                                            Serratia marcescens                                                                         Tc.sup.R                                                        Pseudomonas putida                                                                          Rif.sup.R                                                       Bacillus subtilis BD170.sup.4)                                                              trpC2, thr-5                                                    ______________________________________                                                                     Coordinates of                                                                parB insert                                      Plasmid                      (cf. FIG. 1)                                     R1drd-19                                                                      pSGS8.sup.5)  pBR322.sup.-, Trp.sup.+, Ap.sup.R                               pBOE93        RSF1010, Kan.sup.R                                              pPR95         R1, +(hok.sup.+, sok.sup.+)                                                                  -300-+580                                        pPR311        R1, +(hok.sup.+, sok.sup.+)                                                                   +1-+580                                         pPR633        pBR322, + (hok.sup.+, sok.sup.+)                                                              +1-+580                                         pPR634        pBR322, - (hok.sup.+)                                                                        +194-+580                                        pPR341        pBR322, - (hok.sup.+)                                                                        +268-+580                                        pPR171        pBR322, -      -300-+288                                        pPR154        pBR322, - (sok.sup.+)                                                                        -300-+330                                        pKG634        pBR322, - (hok.sup.+)                                                                        +194-+580                                        pKG341        pBR322, - (hok.sup.+)                                                                        +268-+580                                        pKG171        pBR322,        -300-+288                                        pPKL100       pBR322, Ap.sup.R, Tet.sup.s                                                                  +268-+580                                        pPKL8         pBR322, Ap.sup.R                                                pJK3-1.sup.6) pBC16, pBR322, Tet                                              pSI-1.sup.7)  pUB110, pBR322, Cat                                             ______________________________________                                         .sup.1) M. J. Casabadan, S. N. Cohen, J. Molec. Biol. 138, 1980, p. 179.      .sup.2) R. Simon, Biotechnology, November 1983.                               .sup.3) J. Grinsted, J. R. Saunders, L. C. Ingram, R. B. Sykes. M. N.         Richmond, J. Bacteriol. 110, 1972, p. 529.                                    .sup.4) Dubnan & Cirigliano, J. Bacteriol. 117, 1974, p. 488                  .sup.5) G. Skogman, J. Nilsson, P. Gustafsson, Gene 23, 1983, p. 105.         .sup.6) Kreft et al., in Molecular Cloning and Gene Regulation in Bacilli     eds. A. T. Ganesan et al., Academic Press, 1982, p. 145.                      .sup.7) A slight modification of pAIQ25 described in Yansura and Henner,      Proc. Natl. Acad. Sci. 81, 1984, p. 439; obtained from Henner.           

Purification of chromosomal DNA

Total DNA was extracted from bacteria as follows. Cells were harvestedby centrifugation, washed twice in 1×TEN buffer (TEN=10 mM TRIS (pH7.5), 1 mM EDTA, 0.1M NaCl) and resuspended in 1/10th volume of TENcontaining 1 mg/ml lysozyme. Following incubation at 37° C. for 30minutes, the protoplasts were lysed by addition of sodium dodecylsulphate to a final concentration of 1%, and proteinase K was added to0.25 mg/ml. The Iysate was incubated at 37° C. for 2 hours andsubsequently extracted twice with buffered phenol and three times withchloroform. Sodium acetate was added to 0.3M and the DNA wasprecipitated by addition of 1 volume isopropanol. The precipitate waswashed several times in 96% and 80% ethanol. Finally, the DNA wasdissolved in 1 mM TRIS, 1 mM EDTA.

Total DNA from Tetrahymena thermophila BVII was prepared according toNielsen, H. and Engberg, J.: Biochim. Biophys. Acta 825, 1985, pp.30-38. Macronuclei from Tetrahymena thermophila BVII were isolated(Cech, T. R. et al.: Cell 27, 1981, pp. 487-496) and DNA extracted(Manitis et al., 1982, op.cit., pp. 280-281). rDNA from Tetrahymenathermophila BVII was prepared as described by Engberg, J. et al.: J.Mol. Biol. 104, 1976, pp. 455-470.

Chloroplast DNA from Pisum sativum was isolated according to Bookjans,G. et al.: Analyt. Biochem. 141, 1984, pp. 244-247.

Embryonic liver tissue from a 7-weeks legal abortion was minced inphysiological saline and the DNA was prepared according to Maniatis etal., 1982, op.cit., pp. 280-281. In a similar manner, DNA was isolatedfrom a tumor biopsy from a case of neuroblastoma; the isolated DNA wasfound to contain a several hundred-fold amplified chromosomal regionand, correspondingly, the tumor cells were found to contain numerousextrachromosomal mini-chromosomes by microscopy of mitotic cells.

Isolation of DNA fragments for radioactive labelling

100 micrograms of pPR95 and pBD2724 DNA were digested with EcoRI andEcoRI and HindIII, respectively. The fragments were separated byelectrophoresis through a 1% agarose gel in Tris-borate buffer at 5volts per cm for 3 hours. The desired fragments were isolated byelectroelution onto an NA45 membrane (Schleicher & Schull) according tothe manufacturer's recommendations. Following recovery of the fragmentsby elution of the filter in 1.5M NaCl at 65° C., the fragments wereagain subjected to purification by agarose gel electrophoresis and NA45membrane recovery from the gel.

Agarose gel electrophoresis

The DNA was cleaved with the appropriate restriction endonucleasesaccording to the recommendations given by the manufacturers. Forcellular DNA, 10 units per microgram of DNA was used. The incubationtime was 3 hours at 37° C. The generated DNA fragments were separated byelectrophoresis through 0.7% or 1% agarose gels in a Tris-acetate bufferat 0.8 volt per cm for 18 hours and visualized by ethidium bromidestaining.

A molecular weight marker was prepared as follows: wt λ DNA wasrestricted with HindIII and end-labelled by means of the Klenowpolymerase from Boehringer, Mannheim, as recommended by themanufacturer, in the presence of α-32P-dCTP plus non-radioactive dATPand dGTP. When used as a molecular weight marker, an amount ofTetrahymena macronuclear DNA was added corresponding to the DNA load ofthe test lanes.

Transfer of DNA fragments from gel to nitrocellulose filter

Following partial depurination in 0.25N HCl for 15 minutes at roomtemperature, denaturation of DNA in the gel, neutralization andsubsequent transfer of DNA from gel to a BA85 (Schleicher & Schull)nitrocellulose filter was carried out as described in Maniatis et al.,1982, op.cit., pp. 280-281. Completeness of transfer was assured byethidium bromide staining of the gel after transfer.

Preparation of radioactively labelled probe

0.3 microgram of the 900 bp parB fragment and 0.3 microgram of the 300bp relB-orf3 fragment were radioactively labelled by nick-translation(Maniatis et al., 1982, op.cit.) using 0.25 micromolarα-32P-deoxycytidine triphosphate (3000 Ci per mmol). The unincorporatedradioactive precursor was removed by means of repeated ethanolprecipitations. To each preparation were added 100 micrograms of E. colitRNA as carrier.

The specific activities of the probes were 2-3×10⁸ and 4-5×10⁷ dpm permicrogram of parB and relB-orf3 fragment, respectively.

Hybridization

Filters containing DNA transferred from agarose gels were preincubatedin plastic bags with the hybridization solution (10 ml per 120 cm²) for18 hours at 37° C. with constant shaking. The hybridization solution wasmodified from Wahl et al., Proc. Natl. Acad. Sci. 76, 1979, pp.3683-3687 and contained 38% deionized formamide, 0.75M NaCl, 50 mMsodium phosphate and 10×Denhardt's solution (50×Denhardt's solution is0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll).

Following preincubation, the denatured radioactively labelled probeswere added to appropriate filters. In experiments employing the parBprobe, the concentration of fragment during hybridization was 3 ng/mlwhile the relB-orf3 probe was used at a concentration of 1.3 ng/ml toobtain equimolar concentrations of complementary sequences in the twosituations.

Hybridization was carried out at 37° C. with gentle shaking for 19hours.

The hybridized filters were washed once for 20 minutes at roomtemperature in 0.4×washing buffer, and finally twice for 30 minutes at60° C. in 4×washing buffer. The washing buffer contained 0.6M NaCl, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.5.Autoradiography was performed using X-ray films and intensifyingscreens. Exposure times are indicated in the description of the figures.

The term "filter hybridization analysis" is used in the Examples todenote the following sequence of operations: agarose gel electrophoresisof DNA fragments, transfer of the fragments to nitrocellulose filters,hybridization with the appropriate radioactively labelled probe, filterwashing, and autoradiography of the filter following washing. The datashown in the Examples represent autoradiograms obtained by filterhybridization analysis.

The term homology is used here to denote the presence of any degree ofcomplementarity between a given probe and the nucleic acid species beinganalyzed.

The degree of homology is expressed as the fraction of complementarybases in a duplex nucleic acid molecule formed between a given probe andthe nucleic acid species being analyzed.

The minimum degree of homology which is detectable is a function of theexperimental conditions exployed during hybridization and ofcharacteristics of the probe and the nucleic acid species beinganalyzed.

The degree of homology between the probe DNA and a filter-bound DNAspecies was estimated from the intensity of the actual hybridizationsignal compared to the signal intensity observed for a 100% homologousfilter-bound sequence under the same conditions.

The intensity of the hybridization signal depends primarily on the rateof hybridization and the number of filter-bound DNA molecules present inthe specifically hybridizing band. The rate of hybridization is mainlydetermined by the concentration of complementary sequences duringhybridization, the ionic conditions, the temperature and the degree ofhomology between the probe DNA and the filter-bound molecules. The rateof hybridization decreases by the presence of non-complementarysequences (Bonner, T. I. et al., J. Mol. Biol. 81, 1973, p. 123) whichdecreases the thermal stability of the duplex DNA; 1% mismatch betweenprobe and filter-bound DNA results in a decrease in thermal stability of1 degree (Maniatis et al., 1982, op.cit., p. 388). The hybridizationconditions therefore determine which level of mismatch will still yielda detectable signal. It should be noted that the conditions employed inthe present work did not lead to saturation of the filter-bound DNA withprobe.

The present set of conditions for hybridization and filter subjects DNAduplexes to a temperature which is 40° C. below the mean meltingtemperature of perfectly matched duplex DNA in the same ionicenvironment, i.e. the conditions allow the detection of signals fromduplexes containing a high degree of non-pairing bases. The formula usedin these calculations is discussed in Beltz, G. A. et al., Meth.Enzymol. 100, 1983, pp. 266-285.

It is estimated that the conditions employed detect 100% of the maximumhybridization signal obtained from duplexes with from 100% down to 80%homology while the signal from a 60% homologous duplex is 50% of theabove maximum intensity, cf. above. Duplexes with lower homology than60% will yield still weaker signals.

For duplexes with extensive mismatch, a signal maybe detectable if theexposure time of the autoradiogram can be prolonged or if the number ofcopies of filter-bound complementary molecules can be increased.

EXAMPLE 1

Deletion mapping of the parB region (cf. FIG. 1)

Construction of pPR95 (FIG. 2)

The construction of pPR95 was done in the following way: plasmid pOU93(Gerdes et al., J. Bacteriol. 161, 1985, pp. 292-98) is a pBR322derivative containing the parB PstI fragment derived from the EcoRI-Afragment of plasmid R1 (FIG. 1). The PstI fragment is convenientlydivided into smaller fragments by the restriction enzyme RsaI as shownin FIG. 1. By conventional cloning procedures, the largest RsaI fragment(880 bp) was inserted into the SmaI site of the pBR322 derived cloningvector pHP34 (Prentki et al., Gene 17, 1982, pp. 189-96), resulting inpPR13. The SmaI site of pHP34 is flanked by two EcoRI sites andtherefore the inserted 880 bp RsaI fragment was converted to a 900 bpEcoRI fragment. The so generated 900 bp EcoRI fragment of pPR13 wascloned into the unique EcoRI site of the miniR1 derivative pOU82(International Patent Application No. PGT/DK83/00086, Publication No. WO84/01172), resulting in pPR95. A drawing of pPR95 is presented in FIG.2.

Plasmid pOU82 is unstably inherited due to the lack of any partitioningfunction (International Patent Application No. PCT/DK83/00086,Publication No. WO 84/01172), and has a loss frequency on the order of10⁻² per cell per generation.

On the other hand, pPR95 is very rarely lost and is characterized byhaving a loss frequency of less than 10⁻⁴ per cell per generation(measured as described in International Patent Application No.PCT/DK83/00086, Publication No. WO 84/01172), which is thecharacteristic loss frequency of parB⁺ miniR1 derivatives. Thus, it maybe concluded that the complete parB region is located on the 880 bp RsaIfragment as judged by the ability of the fragment to stabilize miniR1replicons.

The fine mapping of the parB region was carried out as follows: pPR95was restricted with BamHI, treated with exonuclease Bal31, and ligated.Before ligation, BamHI oligonucleotide linkers were added. Thistreatment resulted in a series of deletion derivatives covering theleft-hand part of the parB region. The extension of the deletions wasdetermined by size fractionation of DNA fragments on agarose gels afterthe DNA had been treated with the restriction enzymes EcoRI and BamHI.Subsequently, the precise insertion of the BamHI oligonucleotide linkerswas determined by nucleotide sequencing as described by Maxam andGilbert (Meth. Enzymol. 65, 1980, pp. 499-566). In this way, a verydetailed mapping of the region was obtained. Furthermore, the ParBphenotype (determined as described in Materials and Methods) for eachplasmid derivative was analyzed. Deletion from pPR95 of the sequenceextending from -320 to 0 (cf. FIG. 1) resulting in pPR311 did not changethe ParB⁺ phenotype. Thus, the remaining 580 bp BamHI-EcoRI fragment inpPR311 must contain the complete parB region. Deletion from left furtherinto the region completely abolishes the stabilizing activity.

Deletions into the right part of the 580 bp parB⁺ fragment of pPR311(cf. FIG. 1) resulted in loss of ParB⁺ phenotype, so the parB regionextends to a position close to this end of the fragment.

EXAMPLE 2

Nucleotide sequence of the parB region (cf. FIG. 3)

The nucleotide sequence of the minimal parB region, which is presentedin FIG. 3, was obtained using the chemical degradation method asdescribed by Maxam and Gilbert (Mech. Enzymol. 65, 1980, pp. 499-566).In the following, a detailed description of the essential biologicalinformation in the nucleotide sequence of the parB region is presented.

The sequence of the minimal parB region of 580 bp as defined in Example1 (cf. FIG. 1) is depicted in FIG. 3. The central and left-hand parts ofthe region are very rich in dyad symmetries. The 580 bp contains threeopen reading frames consisting of more than 50 codons. The start andstop codons of these reading frames are indicated in FIG. 3. The readingframe starting at position +304 and ending at +460 is preceded by a DNAsequence (5'-AGGA-3') resembling the E. coli ribosome binding site(Shine and Dalgarno, Nature (London) 254, 1975, pp. 34-38), which isknown to act as recognition site for ribosomes initiating translation ofmRNA. The polypeptide product of this reading frame is shown below theDNA sequence in FIG. 3.

EXAMPLE 3

Functions expressed from the parB region (cf. FIGS. 4 and 5)

A series of plasmids was constructed from which conditional expressionof the putative genes (as indicated from the sequence) in the parB⁺region was obtained through insertion of a fragment carrying the λpRpromoter and the λcI857 gene. The positions of these insertions areindicated in FIG. 1. The λpR temperature inducible promoter system waschosen because the regulator gene for the λpR promoter (the cI857 gene)as well as λpR are located on a single BglII-SalI restriction fragment;furthermore, the cI857 allele of the λ repressor gene makes the insertedpromoter inducible at high temperature (42° C.), but silent or nearsilent at low temperature (30° C.). The BglII-SalI fragment of pOU82 wasinserted into plasmids pPR634 and pPR341 by conventional cloningprocedures yielding plasmids pKG634 and pKG341, respectively (cf.Materials and Methods).

At 30° C., cells harbouring pKG634 and pKG341 grow normally; however,induction of λpR (at 42° C.) results in rapid killing of the host cells.

FIG. 4 shows the killing kinetics (viable counts) and growth measured asOD450 after a shift to 42° C. of strain JC411(pKG634). Viable countsdecrease rapidly (half life of 2.5 minutes) and the increase of OD450stops. The presence of a λpR promoter transcribing the parB region inthe opposite direction (pKG171) has no effect on cell growth andviability (FIG. 4, control).

Microscopic examination (phase contrast) of the cells (JC411/pKG634)after heat induction of the αPR promoter showed that the cells changedmorphology: Most of the material apparently condensed in zones, leavingthe rest of the cell transparent. An illustration of this is shown inFIG. 5, in which both normal and changed cells are present. The cellshaving the characteristic parB induced appearance are termed "ghost"cells in the following.

Since the λpR-promoter fragment was inserted immediately upstream of thestart of a 52 amino acids open reading frame (cf. Example 2), thisstrongly suggests that the 52 amino acids polypeptide encoded by theopen reading frame starting at position +304 (FIG. 3) is responsible forthe cell killing, and consequently, this gene is termed hok (hostkilling) in the following.

EXAMPLE 4

Suppression of the host killing effect expressed by hok (cf. FIG. 6)

A gene from which a highly toxic product is expressed must obviously beregulated. Therefore, it was assumed that the regulator of hok was alsoencoded by the parB⁺ region. In a first attempt to characterize thisregulatory loop, the fragment of pKG634 containing λcI857 upstream ofthe hok gene was inserted into a mtni-F plasmid, resulting in pF634.FIG. 6 represents the induction of killing of JC411 (pF634) which showsthat the killing occurs somewhat slower and less efficiently than in thecase of pKG634 in accordance with the low copy number of F compared topBR322.

A second parB⁺ plasmid (pPR633) was subsequently transformed into strainJC411 (pF634) and the induction experiment repeated with this doubleplasmid strain. As seen in FIG. 6, the parB⁺ region present in transfully suppresses the transcriptional activation of the hok gene. Thus,the parB⁺ region encodes a suppressor of host killing (the sok gene).

Employing this experimental design as an assay, the sok gene was mappedin the following way: Double plasmid strains containing pF634 and one ofthe deletion derivatives pPR634, pPR341, pPR154, or pPR171,respectively, were constructed, and by following the growth pattern ofthese strains at 42° C., the Sok phenotype of the deletion derivativeswas determined by measuring growth after the temperature shift. Theanalysis of these deletion derivatives showed that the plasmids pPR634and pPR154 express Sok activity, whereas the plasmids pPR341 and pPR171express non-detectable levels of Sok activity.

The plasmids were also tested for the incompability phenotypecharacteristic for parB⁺ (cf. International Patent Application No.PCT/DK83/00086, Publication No. WO 84/01172), and it was found thatplasmids expressing Sok activity also exert parB specificincompatibility, whereas plasmids which are Sok⁻ as described above donot exert incompatibility. Thus, the parB incompatibility reactionrepresents an assay for Sok activity.

In a manner similar to that described for the hok gene, the regionrequired for sok gene activity has been further narrowed down. One ofthe sok⁻ derivatives used in the mapping procedure, pPR171, contains theparB⁺ region extending from coordinate -300 to +288 (FIGS. 1 and 3). Arestriction fragment containing the λcI857 and λpR was inserted intopPR171 in such a way that the λpR promoter reads into the sok region ofthe plasmid, resulting in pKG171 (cf. Materials and Methods).

Plasmid pKG171 was transformed to strain CSH50 containing pOU94. PlasmidpOU94 is a lac⁺ parB⁺ p15 derivative which is completely stablyinherited due to the presence of the parB⁺ region on the plasmid.Introduction of other parB⁺ plasmids into that strain results indestabilization of pOU94 due to the incompatibility expressed fromparB⁺. At 30° C., the presence of pKG171 did not result indestabilization of pOU94 to any significant extent, whereas a cleardestabilization was detected at 42° C. Therefore, transcription fromright to left into the parB⁺ region of pKG171 results in activation ofthe incB region (i.e., the sok gene).

The results described here further narrows down the sok gene which musttherefore be located between +194 (pPR634) and +288 (pPR171). Also, itis indicated that the sok gene promoter reads from right to left(opposite of hok gene transcription) and is located at least partly inthe region between +288 (pPR171) and +336 (pPR154). A putative -10sequence (TATCCT) is located at position +262 and a -35 sequence(TTGCGC) is located at position +285 (FIG. 3) (Hawley and McClure,Nucleic Acids Res. 11, 1983, pp. 2237-2255). It is assumed that thesesequences constitute the promoter of the sok gene.

EXAMPLE 5

Discovery of an E. coli chromosomal homologue of R1 parB (cf. FIG. 8a)

Since plasmid evolution has involved an extensive exchange of geneticinformation between bacterial chromosomes and freely replicating DNAmolecules, the chromosomal DNA of E. coli was analyzed for possibleancestral sequences to the R1 parB sequences.

In lane 6 in FIG. 8a, total EcoRI-restricted DNA from plasmid-free E.coli JC411 was analyzed by filter hybridization to a parB probe, cf.Materials and Methods. A fragment of 20 kb is seen to yield a ratherweak, but definite signal which can also be detected in other lanescontaining E. coli DNA if exposed for the same time (lanes 4, 5). Thechromosomal sequence is estimated to be approximately 55% homologous toparB. The chromosomal sequence is named par1 in the following.

A major question is of course to which extent the finding of homology atthe level of the nucleotide sequence also reflects similarity infunction of the products encoded by the homologous regions, an aspectwhich will be further dealt with in Example 6.

EXAMPLE 6

Genetic organization of an E. coli chromosomal homologue of R1 parB andits functional relationship to R1 parB (cf. FIG. 7)

The hok gene of plasmid R1, defined in Example 3, codes for apolypeptide of 52 amino acids. The amino acid sequence of the hok geneproduct was compared to a large number of known protein sequences.Surprisingly, a polypeptide of 51 amino acids encoded by the relB-orf3gene of the E. coli relB operon (Bech et al., The EMBO Journal 4, 1985,pp. 1059-1066) showed significant homology to the hok product. The aminoacid sequences of the two homologous proteins are presented in FIG. 7,which shows that 42% (22) of the amino acids are identical in the twoproteins. For 17% (9) of the amino acids the changes are conservative,meaning that one amino acid has been replaced with an amino acid ofsimilar chemical characteristics (i.e. hydrophobicity, charge, etc.),resulting in an overall homology of 61%. Especially the charged aminoacids are well conserved as are the cysteine residues at positions 16and 31 (FIG. 7).

The DNA sequences of the hok gene and of relB-orf3 were also compared asshown in FIG. 7. The coordinates used in the following are parB⁺sequence coordinates as in FIG. 3. From coordinates +290 to +460, thereis 55% homology between the two sequences. It appears from FIG. 7 thatthe conserved region includes nucleotides upstream and downstream of theprotein coding sequence located from +304 to +460. The conservation ofbases outside the coding region indicates that regulatory features ofthe two genes have also been at least partly conserved.

To show that the sequence homology reflects similarity in function, aplasmid carrying the .English Pound.pR promoter fragment upstream of therelB-orf3 gene was constructed (cf. the description of an analogous typeof construction used in mapping the hok gene in Example 3).

When λpR mediated transcription into relB-orf3 is induced, a rapidkilling of the cells is observed with a kinetics similar to thatobserved for bacteria containing plasmid pKG341 as described in Example3. Simultaneously, all the cells in the culture are transformed into thehok characteristic "ghost" cells (cf. FIG. 5).

Thus, there is a striking homology between the hok gene of plasmid R1and the relB-orf3 of the E. coli relB operon both at the structural andfunctional level.

EXAMPLE 7

parB homologous sequences on various plasmids (cf. FIG. 8a)

Filter hybridization analysis of total, EcoRI-restricted DNA from anumber of strains of E. coli harbouring various plasmids was carried outusing the parB probe (lanes 1-5 in FIG. 8a).

The plasmid R1drd-19 is a member of the R1 plasmid family from which theparB probe was originally cloned. R1drd-19 is present at two copies perbacterial genome. EcoRI-restricted total DNA from E. coli 1005/R1drd-19is analyzed in lane 1. A strongly hybridizing fragment of 19.5 kb isseen, the size of which is consistent with the genetic mapping of theparB function to the 19.5 kb R1 plasmid (International PatentApplication No. PCT/DK83/00086, Publication No. WO 84/01172).

The plasmid R100 is closely related to R1 carrying a transposableelement, Tn10, within the region equivalent to the 19.5 kb EcoRI-Afragment of R1. The transposon contains the recognition sequence forEcoRI and, consequently, a further EcoRI site is introduced into theR1-like EcoRI-A fragment splitting this into the two EcoRI-A and EcoRI-Dfragments of R100 (Miki et al., J. Bacteriol. 144, 1980, pp. 87-99).These two EcoRI fragments of R100 both contain sequences found byheteroduplex mapping to be homologous to sequences present of the Ffactor (Sharp et al., J. Mol. Biol. 75, 1973, p. 235). A stronglyhybridizing fragment of 12.8 kb is seen in lane 2, FIG. 8a, therebymapping the parB region of R100 to the EcoRI-D fragment of R100, withinthe center of the region of homologybetween R1 and R100, and F.

This localization of parB within the F homology region of R100 promptedthe search for parB-like sequences on plasmids belonging to theincompatibility group, IncFI.

EcoRI-restricted, total DNA from B210/R386, an E. coli strain harbouringthe IncFI plasmid K386, was analyzed by filter hybridization using theparB probe (lane 3, FIG. 8a).

The plasmid R386 which belongs to the same incompatibility group as Fwas found to give a parB hybridization signal corresponding to an EcoRIfragment of 19.5 kb. Since this plasmid is present at 0.5-1 copies pergenome, the finding of a signal of approximately one third of the R100signal (lane 2, FIG. 8a) suggests that the degree of homology between R1parB and the R386 parB-like sequences is 55-60%.

The search for parB-related sequences was extended to otherincompatibility groups. The plasmid RP1, which belongs to theincompatibility group IncP, was analyzed.

With the parB probe, total, EcoRI-restricted DNA from 1005 (RP1) yieldsa hybridization signal corresponding to the EcoRI-linearized plasmid(lane 4, FIG. 8a). In addition, a hybridizing band of 20 kbcorresponding to par1 is seen, which was discussed in Example 5.

Since RP1 is adapted to stable maintenance in a broad range ofgram-negative bacterial hosts, the finding of parB-related sequences onRP1 opens the possibility that maintenance systems analogous to the R1parB system, which is operative in E. coli as well as in Pseudomonasputida (Example 11), may function in a multitude of bacterial hosts.

Yet another plasmid, R6-K (IncX incompatibility group), was found tocarry sequences with approximately the same hybridizationcharacteristics as RP1 as evidenced by the presence of a 25 kb EcoRIfragment of R6-K hybridizing the parB probe (lane 5, FIG. 8a).

The low copy number plasmid F has been analysed in some detail in orderto determine whether the presence of R1 parB hybridizing sequencesreflect the existence of a stabilization mechanism related to that of R1parB.

Two plasmid stabilization functions have been identified within thegenome of F and the corresponding genes (sop (Ogura and Hiraga, Cell 32,1983, pp. 351-360) and ccd (Ogura and Hiraga, Proc. Natl. Acad. Sci. USA80, 1983, pp. 4784-4788)) have been located to the EcoRI fragmentspanning the map positions 40.3 to 49.5.

Filter hybridization analysis of total DNA from E. coli 1005 harbouringF showed that R1 parB-related sequences were present on a 10.7 kb EcoRIfragment of F (map position 49.5 to 60.2) and further hybridizationanalyses of 1005(F) DNA digested with EcoRI and/or BamHI mapped thesesequences to a 4.5 kb BamHI-EcoRI fragment extending from map position55.7 to 60.2. This indicates the existence of a third plasmidstabilizing function within F.

The region of F hybridizing the R1 parB probe was subsequently clonedinto a bacteriophage λ vector. EcoRI-digested DNA from 1005(F) wassize-fractionated by preparative agarose gel electrophoresis andfragments of 9.5 to 12 kb were recovered by electroelution from the gel.The fragments were ltgated to the EcoRI sites of the left and right armsof λL147 and packaged in vitro to yield infectious phages (cf. Maniatiset al., Molecular Cloning, Cold Spring Harbor, N.Y., 1982, p. 256) whichwere then used to infect E. coli LE392. Recombinant phages carrying theR1 parB-related sequences were identified by plaque hybridization.

From a recombinant phage carrying the 10.7 kb EcoRI fragment whichincludes the R1 parB-related sequences, the fragment was isolated andinserted into pUC8 at the EcoRI site. In one resulting plasmid, pNL1,the insert is so oriented that cleavage of pNL1 DNA with BamHI resultsin excision of a 4.5 kb fragment carrying the R1 parB hybridizingsequences.

The 4.5 kb BamHI fragment from pNL1 was isolated and the regionhybridizing the R1 parB probe was mapped to an RsaI fragment of 870 bpby filter hybridization analysis. The 870 bp RsaI fragment was isolatedand inserted into the SmaI site of M13mp9. A number of recombinantphages carrying the R1 parB related sequences on a 870 bp insert wasidentified by plaque hybridization. The nucleotide sequence of theinserted DNA was analysed according to Sanger et al., Proc. Natl. Acad.Sci. USA 74, pp. 5463-5467.

The nucleotide sequence of part of one of the recombinant phages,mpNL12, comprises 402 bases extending from the RsaI site and thissequence is 90% homologous to the region from +178 to +580 of the R1parB sequence (FIG. 3). All essential features of the R1 parB region arealso found in the F-derived sequence: (1) an open reading frame encodinga protein of 50 amino acids is present corresponding to the R1 hok gene,(2) the ribosome binding site of R1 hok is conserved, (3) the regioncorresponding to the 3' non-translated part of R1 parB mRNA, which isbelieved to be essential for hok mRNA stability, is highly conserved(90% homology), and (4) the putative -10 and -35 regions of R1 sok arealso conserved.

The open reading frame within the F-derived sequence codes for a proteinof 50 amino acids which differs only slightly from the R1-specified hokprotein. Firstly, two codons in R1 hok have been deleted, namely val-15and Ser-29. Secondly, two conservative substitutions have occurred,namely leu-16 to val and his-39 to tyr.

Evidently, the R1 hok gene and the related sequences on F derive from acommon ancestral sequence and, furthermore, the conservation of a codingregion corresponding to R1 hok strongly suggests that the encodedprotein is involved in the stabilization of F.

To test for plasmid stabilizing properties of the F-derived sequence,the 4,5 kb BamHI fragment from pNL1 which carries the F hok-likesequences was inserted into pJEL82, a low copy number plasmid with aloss frequency of 10⁻² per generation (cf. PCT/DK83/00084, PublicationNo. WO84/01171). The resulting plasmid, pJEL82/F, as well as pJEL82 wastransformed into E. coli HB101. Cultures of the two strains were grownfor 16 hours without selection pressure and the fraction ofplasmid-containing cells (Ap^(R)) was determined. The result was asfollows:

    ______________________________________                                               plasmid                                                                              % Ap.sup.R  cells                                               ______________________________________                                               pJEL82 36.5                                                                   pJEL82/F                                                                             98.4                                                            ______________________________________                                    

It was therefore concluded that the 4.5 kb BamHI fragment carrying R1parB related sequences exerts a plasmid stabilizing effect. If thestabilization is due to the presence of the hok-like gene within the Ffragment, the emergence of ghost cells would be expected in cultures ofcells harbouring pJEL82/F grown without selection pressure, cf. Example3. An overnight culture of cells containing pJEL82/F was found tocontain approx. 5% ghost cells indistinguishable from R1 hok inducedghost cells.

In case of F, the demonstration of sequences related to R1 parB byfilter hybridization thus reflects the existence of a functionallysimilar plasmid stabilization mechanism.

EXAMPLE 8

Stepwise hybridization as a strategy for the detection of repliconstabilizing sequences homologous to parB related sequences (cf. FIG. 8b)

The conditions of hybridization determine the level of homology betweena probe and a filter-bound DNA species required to yield a detectablesignal, cf. the discussion in Materials and Methods. Consequently,filter-bound sequences may exist which remain undetectable with thegiven probe under the given set of hybridization conditions but whichmay nevertheless encode a hok-like activity, cf. the discussion ofhomology versus function in Materials and Methods. This is illustratedin the following experiment.

As described in Example 6, the relB-orf3 represents a chromosomalhomologue of R1 parB based on the sequence comparison data and thefunctional similarity of hok and relB-orf3. The relB-orf3 and flankingsequences, as present in plasmid pBD2724, was used as probe in a filterhybridization analysis of E. coli chromosomal DNA.

Plasmid pBD2724 is a pBR322 derivative containing a HincII-MluI fragmentfrom the relB operon of E. coli comprising the relB-orf3 coding sequence(coordinates 1070-1350 according to Bech et al., op. cit.).

In lane 3, FIG. 8b, total EcoRI-restrtcted DNA from plasmid-free E. coliis analyzed by filter hybridization using the relB-orf3 probe. Inaddition to the 20 kb hybridizing fragment likely to represent theabove-identified par1 sequence (Example 6), yet another hybridizingfragment of 16 kb is detected. Since the intensity of the latter isgreater than the intensity of the 20 kb signal, the 16 kb EcoRI fragmentmust span the E. coli relB-orf3 gene used as hybridization probe, i.e.the intensity of the 16 kb signal provides a reference from whichdegrees of homology can be estimated. The intensity of the par1hybridization signal, which is approximately 3/4 of the relB-orf3signal, suggests that par1 is approximately 65-70% homologous torelB-orf3. Since the 16 kb relB-orf3-carrying fragment is not detectedwith the parB probe (lane 6, FIG. 8a), R1 parB is 50% or less homologousto relB-orf3.

In Example 5 it was found that the parB probe detects the 20 kbpchromosomal homologue but not the 16 kbp homologue representing therelB-orf3 according to the above data. Since, as described in Example 6,the latter exerts hok-like activity when expressed, it can be assumedthat the par1 will also express hok-like activity or sok-like activityand/or both activities when properly expressed.

The relB-orf3 fragment was used as a probe in filter hybridizationanalysis of E. coli harbouring plasmid R100, and R386, both of whichcontain R1 parB-like sequences (FIG. 8a, lanes 1 and 2). Under thepresent set of hybridization conditions, the relB-orf3 probe did notdetect these sequences (FIG. 8b, lanes 1 and 2) since only the 20 kbpar1 and the 16 kb relB-orf3 carrying fragment are seen to hybridize theprobe, thereby indicating that the absence of hybridization between aprobe from a region expressing hok or hok-like activity and a givenDNA-species does not preclude that the DNA-species in question can exerthok-like activity if properly expressed. Consequently, the finding ofhomology between the DNA-species in question and a region expressing hokor hok-like activity strongly suggests that the DNA-species in questionwill exert hok or hok-like activity if properly expressed.

The above data therefore reveal a useful strategy in searching forregions exerting hok/sok-like activities: A probe representing a regionof nucleic acid comprising hok or hok-like genes (e.g. R1 parB) is usedto detect homologous sequences (e.g. par1) within the genome in question(e.g. chromosomal or plasmid DNA) which are subsequently tested for hokor hok-like activity (as done for the relB-orf3 region) in the properexperimental settings, and if shown to encode such activity oractivities are next used themselves as probes in a second round ofhybridizations to define novel homologous sequences which may or may notbe related to the probes used in the first round of hybridizations (e.g.R1 parB). This stepwise procedure combining nucleic acid hybridizationand functional assays of the isolated nucleic acid sequences may beadapted as a general strategy to search for genes expressing hok orhok-like activities in genomes increasingly separated from E. coli onthe evolutionary scale.

EXAMPLE 9

Detection of parB related sequences in bacteria (cf. FIGS. 9 and 10)

In the previous Examples, it was demonstrated 1) that sequences relatedto R1 parB are widely distributed among bacterial plasmids isolated fromgram-negative bacteria, and 2) that sequences related to R1 parB arepresent in the chromosomal DNA of E. coli. These findings prompted asearch for sequences related to either R1 parB or one of the chromosomalcounterparts (E. coli relB-orf3) in DNA from a variety of bacteria, aspart of either their chromosomal DNA or of plasmids naturally present inthese organisms.

Filter hybridization analysis of EcoRI-restrtcted DNA from Serratiamarcescens with the R1 parB probe shows intense hybridization to 3fragments of 4.1, 2.9 and 2.5 kb (lane 2, FIG. 9). Only the 4.1 kbfragment also hybridizes the relB-orf3 probe (lane 1, FIG. 10). The parBprobe hybridizes an additional 6 fragments. Two of these signals arestronger than the parB signal derived from the relB-orf3-carrying 6 kbfragment in E. coli DNA (lane 6, FIG. 9). Hybridization of Serratiamarcescens DNA with the E. coli relB-orf3 probe yields a number of weakhybridization signals. It is possible that the strongly hybridizingbands of 2.5, 2.9 and 4.1 kb are derived from plasmid(s) although theagarose gel electrophoresis did not reveal any high copy numberplasmids. Pseudomonas fluorescens was analyzed as a plasmid-free memberof this species. Hybridization of DNA from Pseudomonas fluorescens withR1 parB (lane 3, FIG. 9) shows 8-10 hybridizing fragments, 4 of whichexhibit signals with intensities of approximately 33% of the chromosomalcounterpart of R1 parB (lane 6, FIG. 9). A single of these fragments, ofapproximately 13 kb, probably also hybridizes the E. coli relB-orf3probe (lane 2, FIG. 10). In addition, the relB-orf3 probe identifies 5fragments specifically, although at low signal intensity; two of these,of 5.5 and 5.6 kb, are also seen in DNA from Pseudomonas putida whenthis DNA is analyzed using the relB-orf3 probe (lane 3, FIG. 10). TherelB-orf3 probe hybridizes to an additional 5 fragments in Pseudomonasputida DNA, but none of these fragments are recognized by the R1 parBprobe (lane 4, FIG. 9). In Pseudomonas putida DNA, the parB probedetects approximately 10 fragments of low signal intensity and a singlequite strongly hybridizing fragment of approximately 7.3 kb.

Among gram-positive bacteria, B. subtilis, B. circulans PL236 and twostrains of Lactobacillus were analyzed for the presence of sequencesrelated to either R1 parB or E. coli relB-orf3.

In case of the parB probe, a single quite strongly hybridizing fragmentof 5.2 kb was found in DNA from B. circulans (lane 8, FIG. 9). Very weaksignals were obtained from a few additional fragments of B. circulansDNA. With the relB-orf3 probe, a limited number of hybridizing fragmentswas seen in DNA from B. subtilis (lane 4, FIG. 10), B. circulans (lane5, FIG. 10), and Lactobacillus (lanes 6 and 7, FIG. 10). The number ofrelB-orf3-hybridizing fragments ranged from 6 to 11, and all haveapproximately the same signal intensity. In the Lactobacilli, agarosegel electrophoresis has demonstrated the presence of plasmids suggestingthe possibility that at least some of the hybridizing sequences are ofplasmid origin. A search for plasmids in B. circulans PL 236 has beennegative suggesting that the sequence of B. circulans DNA hybridizingthe R1 parB probe (lane 8, FIG. 9) may be of chromosomal origin.

The above experiments indicate that sequences related to R1 parB and/orto E. coli relB-orf3 are widely distributed among bacterial species, notonly the Enterobacteriaceae from which the probes were derived, but alsothe gram-positive bacteria.

EXAMPLE 10

Detection of parB related sequences in eukaryotic cells (cf. FIG. 11)

A unicellular organism was investigated, namely the ciliate protozoanTetrahymena thermophila, FIG. 11. This organism is characterized by 1) ahigh number of mitochondrial DNA molecules per cell and 2) approximately12,000 copies of ribosomal RNA genes located on self-replicating rDNAmolecules. Neither the R1 parB probe nor the E. coli relB-orf3 probedetect any fragments in DNA prepared from isolated macronuclei (lane 1,FIG. 11). Nor did the probes hybridize to the two EcoRI fragments ofisolated rDNA (lane 3, FIG. 11). Total EcoRI-restricted DNA fromTetrahymena thermophila, which includes mitochondrial DNA, showed twohybridizing fragments, of 6.6 kb and 3.3 kb (lane 2, FIG. 11), with therelB-orf3 probe while the parB probe did not yield any signals. Thehybridizing fragments co-migrated with two EcoRI fragments ofmitochondrial DNA that were readily detectable by ethidium bromidestaining of the gel prior to DNA transfer.

Chloroplast DNA from pea (Pisum sativum) was cleaved with therestriction endonuclease PstI, and 0.125 microgram was analyzed byfilter hybridization using the parB and the relB-orf3 probes (lane 4,FIG. 11). The latter probe hybridizes to a fragment of approximately 16kb.

Finally, two samples of human cellular DNA were analyzed by filterhybridization following EcoRI restriction. The R1 parB probe yielded a(weak) hybridization signal to the neuroblastoma DNA (lane 5, FIG. 11)as well as to the embryonic liver DNA (lane 6, FIG. 11). The highmitochondrial content of liver tissue may indicate that the observedsignal in lane 6, FIG. 11, is derived from human mitochondria. Theneuroblastoma DNA was analyzed since other hybridization analyses hadindicated selective amplification of a small chromosomal region leadingto the presence of extrachromosomal mini-chromosomes ("double minutes");the origin of the hybridization signal in lane 5, FIG. 11, is unknown.

Simultaneously, all the cells in the culture are transformed into thehok characteristic "ghost" cells (cf. FIG. 5).

Thus, there is a striking homology between the hok gene of plasmid R1and the relB-orf3 of the E. coli relB operon both at the structural andfunctional level.

EXAMPLE 11

Construction of a trp-hok fusion

Plasmid pPR341 carries the hok⁺ gene from the parB region without itsnatural promoter (cf. Table 1 and FIG. 3). Plasmid pSGS8 carries the trpoperon on an EcoRI fragment inserted in pBR322. An XhoI-EcoRV fragment(ca. 700 bp) from pSGS8 carrying the trp promoter was inserted byligation in pPR341 digested first with BamHI (this site was madeblunt-ended through a filling-in reaction with Klenow polymerase) andthen with SalI. This insertion placed the trp promoter fragment in suchan orientation that transcription would enter the hok gene. Aftertransformation to MC1000 colonies were selected on LB plates containingampicillin and subsequently, the colonies were tested for growth on A+Bminimal plates containing leucine and ampicillin. In the absence oftryptophan, the trp promoter is induced, and transcription into the hokgene was assumed to be lethal. Therefore, screening was for clones thatdid not grow on the minimal plates. One such clone harbouring a plasmid,p341-1, which was shown by restriction enzyme mapping to have thecorrect insertion, was chosen for further analysis (cf. FIG. 12).

EXAMPLE 12

Growth of cells harbouring p341-1

MC1000 (p341-1) was grown in A+B minimal medium supplied with 0.2%glucose and 1% casamino acids. Casamino acids contain very littletryptophan, so it was expected that at a certain cell density, themedium would be depleted of tryptophan. This situation mimics growth ina natural environment with a limited supply of tryptophan which is arare amino acid. As seen in FIG. 13, the initial growth rate of theplasmid carrying strain (hok⁺) is identical to that of a plasmid-freeMC1000 strain. However, at a cell density of approximately OD₄₅₀ =0.8,growth of MC1000 (p341-1) stops abruptly, indicating induction of thehok gene (verified by microscopic examination of the cells, cf. FIG. 5),whereas the plasmid-free strain keeps growing. Viable counts from MC1000(p341-1) at this point and one hour later show a dramatic reduction inviability (less than 10⁻⁴). In conclusion, the presence of p341-1 in anE. coli K-12 strain makes growth and viability dependent on the presenceof tryptophan in the growth medium. When this amino acid is exhaustedfrom the environment, growth stops immediately and the cells are killed.

EXAMPLE 13

Use of an R1 hok homologue in the construction of a biologicalcontainment system

The F hok gene (cf. Example 7) and the trp promoter were combined togenerate a biological containment system. From pNL1 described in Example7, the 850 bp RsaI fragment hybridizing the R1 parB probe was clonedinto SmaI-restricted M13mp11. A recombinant, mpNL4, was identified inwhich the ribosomal binding site of F hok as well as the coding regionof F hok could be excised as an approximately 300 bp FspI-EcoRIfragment. This approximately 300 bp fragment, the 550 bp EcoRV-XhoIfragment of pSGS8 containing the trp promoter as well as the initialportion of trpE, and the 3.7 kb SalI-EcoRI fragment of pBR322 carryingthe bla gene were ligated to generate pNL7. In this construct, the trppromoter will transcribe into F hok. The plasmid pNL7 has the correctrestriction enzyme pattern, and E. coli HB101 cells transformed withpNL7 show growth inhibition on plates without tryptophan as describedfor p341-1 transformants in Example 12.

The maximum inducible cell death caused by the expression of the F hokgene on pNL7 was determined for E. coli HB101 transformed with pNL7,HB101(pNL7). The cells were grown in Modified A+B (MA+B) which comprisesA+B medium supplemented with thiamine, leucine, proline, 0.4% glucose,1% casamino acids, and ampicillin (50 μg/ml) with further addition oftryptophan at varying concentrations (0-200 μg/ml). The cells were grownto the early exponential phase at which time 100 μg/ml of indolylacrylic acid (IAA) was added which substance competes with tryptophanfor binding to the repressor, thus leading to inactivation of therepressor. The number of viable cells per ml was determined by platingsamples of the IAA-treated cultures onto LB plates with 50 μg/mlampicillin at 30 minutes following IAA induction. E. coli MB101(pBR322)served as a control in these experiments. The maximum killing effect onIAA addition was observed at 5-10 μg/ml tryptophan, the survivingfraction of HB101(pNL7) being 1.4×10⁻⁴. The control cells wereunaffected by IAA addition.

The kinetics of IAA-induced killing of HB101(pNL7) grown in the abovemedium with 5 μg/ml tryptophan is biphasic with an exponential componentfrom 0 to 15 minutes at which time the surviving fraction comprises lessthan 10⁻³, and a second linear phase extending beyond 90 minutes whichfurther reduces the surviving fraction by a factor of 10 or more.

Simulated release experiments were carried out by growing E. coliHB101(pNL7) under conditions leading to depletion of tryptophan andhence to activation of the trp promoter. The cells were grown in MA+Bmedium with either no added tryptophan or supplemented with 5 μg/mltryptophan. OD₆₀₀ and viable cells per ml was followed. The control wasHB101(pBR322).

In one experiment, no tryptophan was present in the growth medium, andas shown in FIG. 14, the OD₆₀₀ of HB101(pNL7) increased exponentiallyfor several hours, albeit at a slower rate than the control culture, butno corresponding increase in cell number was seen. Microscopically, thecell size of pNL7-transformed cells increased during this phase. Sinceviable counts did not drop, it is assumed that a low, but tolerableexpression of F hok took place. Upon depletion of tryptophan, killing ofHB101(pNL7) was observed, the surviving fraction being 2×10⁻³.

In a second experiment, HB101(pNL7) and HB101(pBR322) were grown in MA+Bsupplemented with 5 μg/ml tryptophan. As appears from FIG. 15, the twostrains had identical generation times during the first phase of theexperiment as determined by OD₆₀₀, and in this experiment the increasein OD₆₀₀ in the HB101(pNL7) culture reflected an exponential increase incell number. Thus, the mere presence of the F hok system within E. colidoes not affect cell growth as long as tryptophan is present in thegrowth medium. At the time of tryptophan depletion, the F hok isexpressed due to derepression of the trp promoter resulting in killingof the cells so that the surviving fraction is reduced to 10⁻³ withinone hour.

Using the above construction with F hok, a substantial fraction of cellssurvive induction of expression of the killing function. This might inprinciple be due to one or a combination of several factors: structuralinstability of pNL7, adaptation of HB101(pNL7) to the toxic effect ofthe hok protein, selection of plasmid-free cells in the population, orinsufficient expression of F hok in individual cells due to the combinedeffect of a relatively low level of transcription even in the inducedstate as the trp attenuator is present and selection of cells with lowcopy numbers.

To approach this question, E. coli HB101(pNL7) surviving either 90minutes of IAA induced F hok expression or induction of hok expressiondue to tryptophan depletion following growth with or without exogenouslyadded tryptophan was analysed. The survivors from the depletionexperiments were sampled 1 hour following the deflection of the OD₆₀₀curves in FIGS. 14 and 15. In these three cases, all surviving coloniestested were resistant to 50 μg/ml ampicillin, but survivors from thedepletion experiment in which no exogenous tryptophan had been added(FIG. 14) showed a 50% decrease in cells resistant to 500 μg/mlampicillin. Thus, a continued low-level expression of a hok protein maylead to selection of cells with a low plasmid copy number. 12ampicillin-resistant colonies from each of the three inductionexperiments were grown in LB medium with 50 μg/ml ampicillin to theearly exponential phase at which time IAA was added to 100 μg/ml. Thenumber of viable cells per ml was determined 90 minutes after IAAaddition. In all 36 experiments, the expected killing was observed, thesurviving fraction varying between 10⁻³ and 10⁻⁴. It is thereforeconcluded that the survival of HB101(pNL7) on induction of the F hoksystem*is due to selection of cells in which the level of expression ofhok protein does not exceed a threshold value, e.g. due to selection ofcells with a low plasmid copy number. The efficiency of hok-basedbiological containment systems may therefore be further improved bysubstituting a stronger promoter and/or by increasing the translationalefficiency of the hok mRNA.

EXAMPLE 14

The effect of a λP_(R) promoter on the expression of the hok gene

Treatment of pPR633 with the exonuclease Bal31 resulted in a series ofdeletions in the 5' end of the coding strand of the parB region, two ofwhich are shown in FIG. 16a. The plasmid pPR341 is described in Example3. Plasmid pPR345 covers the +303-+580 region of parB, which onlycontains the reading frame of the hok gene (see FIG. 3). The deletionoffers neither a promoter nor a Shine-Dalgarno sequence to express thehok gene. Cloning of the BglII-SalI fragment containing the λP_(R)promoter and the λ repressor (cI857) into pPR345 restricted with SalIand BamHI resulted in plasmid pKG345 (FIG. 16b). Induction of λP_(R) (at42° C.) resulted in rapid host killing, which showed that pKG345 showedthe hok⁺ phenotype when induced, and that the effect was increasedcompared with previous constructions.

On closer analysis, the construction showed that the cro gene of the λfragment had fused to the hok gene. This resulted in a fusion in whichthe hok gene was expressed from the λP_(R) promoter and the croShine-Dalgarno sequence. The increased host killing effect is thereforedue to a more efficient translation from the cro Shine-Dalgarno comparedwith the natural ribosomal binding site of hok.

FIG. 17 shows the increased killing effect expressed by the cro-hokfusion. The killing kinetics (viable counts) and growth (OD₄₅₀) is shownafter a shift to 42° C. of E. coli strain MC1000 containing eitherpKG341 or pKG345. Increase in OD₄₅₀ stops and viable counts decreaserapidly in both cultures, but the host killing effect of the cro-hokfusion is more distinct compared with hok alone (half life for pKG345 of1 minute).

EXAMPLE 15

Biological containment of a plasmid carrying the trp-hok containmentsystem

Plasmid transfer in natural environments is a highly uncertain riskfactor in connection with recombinant DNA applications. Plasmids wouldtherefore be safer to use if they carried functions that after transferwould induce killing of the new host cell. In an attempt to investigatethe potential of the hok gene product as a killing factor for otherbacterial species, a fusion plasmid of p341-1 and a kanamycin resistantderivative of the mobilizable broad-host-range plasmid RSF1010, pBOE93,was constructed. This hybrid (EcoRI-EcoRI fusion) was transformed to E.coli S 17.1, which harbours a conjugative plasmid RP1 derivativeinserted in the chromosome. In a series of mobilization experiments,p341-1-RSF1010 was transferred from S 17.1 to E. coli 1005, Serratiamarcescens and Pseudomonas putida, respectively. Transfers of pBOE93 andpBOE93 fused with pBR322 were performed as controls.

The results indicated in Table 2 show that all plasmids were transferredwith equal frequencies from S 17.1 to 1005 (it was shown that the 1005(p341-1-RSF1010) transconjugants were killed in the absence oftryptophan).

The vector plasmids were transferred to S. marcescens and P. putida withvery high frequencies, whereas p341-1-RSF1010 was transferred with lessthan a 10⁴ fold lower frequency to both of these bacteria, even iftryptophan was present all the time. Thus, the hok gene product islethal even for the very distantly related P. putida species, and inboth organisms, the E. coli regulatory system for the trp promoter ismissing, although S. marcescens is closely related to E. coli. Thismakes it likely that the great majority of bacteria in the naturalenvironment which have a possibility of receiving E. coli plasmids willbe killed independently of the external concentration of tryptophan whenthe trp-hok fusion is present.

                  TABLE 2                                                         ______________________________________                                        Transfer of p341-trp RSF1010                                                                                     Transfer                                   Donor         Recipient   Selection                                                                              frequency                                  ______________________________________                                        S 17.1 (pBOE93)                                                                             E. coli 1005                                                                              Kan + Nal                                                                              >10.sup.-1                                 (control)     S. marcescens                                                                             Kan + Tc >10.sup.-1                                               P. putida   Kan + Rif                                                                              >10.sup.-1                                 S 17.1        E. coli 1005                                                                              Kan + Nal                                                                              >10.sup.-1                                 (p341-trp RSF1010)                                                                           S. marcescens                                                                            Kan + Tc <10.sup.-5                                               P. putida   Kan + Rif                                                                              <10.sup.-5                                 ______________________________________                                    

EXAMPLE 16

Construction of biological containment systems for grampositivebacteria: identification of hok as a suitable killing function

In the previous Examples, the use of R1 hok as well as homologous genesfor the construction of containment systems has been described for awide range of gramnegative bacteria. However, the widespread use ofgrampositive bacteria in fermentation and the indications thatgrampositive bacteria have a potential use in deliberate releaseproductions call for the development of biological containment systemsfor grampositive bacteria. A prerequisite for constructing containmentsystems similar to those described above for gramnegative bacteria isthat a cell killing function affecting grampositive bacteria can beidentified. Since the R1 Hok protein is toxic in a wide range ofgramnegative bacteria, possible toxic effects of the Hok protein ingrampositive bacteria were investigated.

Preliminary experiments showed that the native promoter and ribosomalbinding site from the R1 hok gene does not promote the expression of hokin B. subtilis.

In order to obtain expression of hok in B. subtilis, the coding sequencefor Hok was inserted into an expression vector containing a promoter aswell as a ribosomal binding site known to be functional in B. subtilis.The plasmid pSI-1 is a modification of pAIQ25 (Yanzura and Henner, Proc.Natl. Acad. Sci. USA 81, January 1984, pp. 439-443) in which theP_(pac-I) promoter and the pentcillase gene have been replaced by aspac-I promoter followed by a synthetic ribosomal binding site(AAGGAGGTGATC) and a polylinker. A gene inserted into the polylinker ofpSI-1 will be expressed if IPTG is added to the growth medium due to thepresent of the lac operator and the lacI gene on the plasmid (Yanzuraand Henner, op.cit.). Before cloning R1 hok into the pSI-1 vector, thehok gene was modified as follows. A double-stranded oligonucleotidecorresponding to the N-terminal Hok coding region and with overhangscorresponding to XbaI (5') and SauIIIA (3') cleavages was synthesized bymeans of a Cyclone™ DNA synthesizer (available from Biosearch Inc., NewBrunswick, USA) using the β-LINK cyanoethyl phosphoramidite synthesismethod. The oligonucleotide differed from the R1 hok sequences at threepositions, one effect being the formation of a HindIII recognition site.

A ligation reaction consisting of the oligonucleotide, a 250 bpSauIIIA-PstI fragment corresponding to the C-terminal portion of the Hokcoding sequence (derived from a pUC9 recombinant from which the hok genecan be excised with various restriction enzymes), and pUC18 restrictedwith XbaI and PstI was used to transform E. coli DH5a (available fromBethesda Research Laboratories, USA), selecting on LB plates containing50 μg/ml ampicillin, 0.004% X-Gal and 1 mM IPTG. From one of the whitecolonies, the plasmid pLK24 was isolated. This plasmid has the expectedphysical map as determined by restriction enzyme analysis. The region ofpLK24 derived from the synthetic oligonucleotide was sequenced in orderto verify the reestablishment of the coding region of the hok gene.

To insert the modified hok gene into pSI-1, the 300 bp hok-carryingXbaI-PstI fragment from pLK24 was isolated and ligated to XbaI and PstIrestricted pSI-1, and the ligation reaction was used to transformcompetent B. subtilis BD170 cells according to the procedure describedin Sadaie and Kada, J. Bact. 153, February 1983, pp. 813-821, withsubsequent selection on LB plates containing chloramphenicol (5 μg/ml).Since this construction positions the ATG start codon of hok at adistance of 11 bp from the synthetic ribosomal binding site of pSI-1,chloramphenicol-resistant colonies were further analysed for inhibitionof growth, the expected phenotype if hok is active in B. subtilis, byplating the transformants on LB plates containing 1 mM IPTG. Growthinhibition was observed for approximately half of the transformantstested. One such transformant, BD170(pLK26), was further analysed.

Plasmid DNA isolated from BD170(pLK26) showed the expected physical map(FIG. 18) and thus appeared to be structurally stable.

In order to test the toxicity of the R1 hok gene product in B. subtilis,BD170(pLK26) and BD170(pSI-1) were grown in LB medium to the earlyexponential phase (OD₆₀₀) at which time IPTG was added to 2 mM. Thisinduces the spac-I promoter of pSI-1. The OD₆₀₀ and viable cells per mlwere determined during the experiment. As can be seen from FIG. 19, thegrowth rates of the two cultures were identical prior to the addition ofIPTG, i.e. the mere presence of the hok gene within B. subtilis does notaffect cell growth. Following addition of IPTG, the OD₆₀₀ of theBD170(pLK26) culture was doubled during the first hour of IPTG treatmentfollowed by a period with no increase in OD₆₀₀ ; this corresponds to thepattern observed for the Hok effect in E. coli. Viable counts decreasedimmediately upon addition of IPTG to the BD170(pLK26) culture (FIG. 20),the surviving fraction being 0.25. No effect of IPTG addition was seenon the control culture. Phase contrast microscopy of BD170(pLK26) onehour after the addition of IPTG showed the appearance of approximately5% of ghost cells. Cells surviving 1.5 hours of IPTG induction of hokexpression were tested for their sensitivity to re-induction with IPTG.All 25 colonies from surviving cells showed growth inhibition on LBplates containing 1 mM IPTG.

It is concluded from these results that the hok gene product is toxic togrampositive bacteria, and hence it can be used to design biologicalcontainment systems according to the principles described in the presentspecification. The finding that the surviving fraction constitutes 0.25does not invalidate this statement since survival seems to be due mainlyto insufficient expression of the hok gene, a feature which may bemodified by, for instance, using a stronger promoter or other standardprocedures.

EXAMPLE 17

A stochastic killing effect obtained by combination of the fim and hoksystems

A stochastic killing effect in E. coli K-12 was obtained by arecombinant plasmid which carried the hok gene in connection with thepart of the fim gene cluster that specifies the periodic expression oftype 1 fimbriae in E. coli.

Plasmid pPKL8 (FIG. 21) carries the 300 bp invertible DNA fragment whichharbours the promoter for the fimA gene. The plasmid further containsthe two regulatory genes fimB and fimE (the "on" and "off" genes,respectively, cf. Klemm et al., Mol. Gen. Genet. 199, 1985, pp. 410-414;Klemm, Embo J. 5, 1986, pp. 1389-1393). Plasmid pPR341 (FIG. 22) haspreviously been described (Gerdes et al., Proc. Natl. Acad. Sci. USA 83,1986, pp. 3116-3120).

pPKL8 was restricted with BglII and BclI. The 3.3 kb BglII-BclI fragmentcontaining the fimB and fimE genes and the invertible promoter regionwas inserted into the BamHI site of pPR341 resulting in plasmid pPKL100(FIG. 23) which was transformed to E. coli K-12 strain MG1000. E. coliK-12 cells harbouring this construct grew normally in LB-medium.However, cultures of such cells showed the presence of 1-2% of ghostcells, many of which were abnormally long (FIG. 24a and b) . This isindicative of a periodic transcription of the hok gene with ensuingkilling of the host.

In order to determine the orientation of the invertible promotersegment, plasmid pPKL100 was digested with the restriction enzymes SnaBIand SacII. The invertible promoter segment contains a unique site forSnaBI, and by studying the sizes of the resulting fragments, theconfiguration of the promoter-containing segment was estimated: A 650 bpSacII-SnaBI fragment results from the "on" configuration and a 350 bpfragment indicates the "off" configuration (see FIG. 23). In the absenceof selection pressure, a 50/50 distribution of plasmids containing thesegment in the "on" and "off" configuration, respectively, is to beexpected as exemplified by pPKL8 (lane B in FIG. 25). However, the samepattern was not seen in the case of plasmid pPKL100 where only the "off"configuration was evident (lane A in FIG. 25). This indicates that cellscontaining plasmid pPKL100 in which the inversional switch is in the"on" configuration are not viable.

EXAMPLE 18

Biological containment based on competition between cells with andwithout a fimA-hok containment system

In order further to elucidate whether the invertible DNA segment inplasmid pPKL100 (cf. Example 17) could be influenced in trans by thegene dosage of fimB and fimE, the following constructs were made tocomplement this plasmid in trans: since plasmid pPKL100 was based on thepBR322 replicon, three plasmids based on the compatible vector pACYC184were constructed, pLP5 carrying the fimB and fimE genes, pLP4 containingthe fimB gene only, and pLP6 containing the fimE gene only.

Plasmid pLP4 was constructed by inserting a 2300 bp BglII-StuI fragmentfrom pPKL10 (Klemm, 1986, op.cit.) into BamHI and EcoRV digested plasmidpACYC184 (see FIG. 26). Plasmid pLP5 was constructed by inserting a 2650bp BglII-SnabI fragment into BamHI and EcoRV digested plasmid pACYC184.Plasmid pLP6 was a HincII deletion derivative of pLP5 (see FIG. 26).Plasmids pLP4, pLP5 and pLP6 were transformed into E. coli MC1000 hostcells already containing pPKL100, which were grown on 0.2% glycerol A+Bmedium supplemented with 10 μg/ml proline, threonine, isoleucine andleucine, 20 μg/ml chloramphenicol and 100 μg/ml ampicillin. The growthrates, as measured by the increase in the optical density of the threecombinations, were as shown in Table 3. The copy number ratio of pBR322to pACYC184 is roughly 4:1, and consequently hosts harbouring theplasmid combination pLP4+pPKL100 have a corresponding 25% increase inthe gene dosage of fimB (which mediates an "off" to "on" configurationof the invertible DNA fragment containing the fimA promoter in pPKL100),as compared to cells harbouring pPKL100 only. On the other hand, cellscontaining the plasmid combination pLP6+pPKL100 have a roughly 25%increase in the amount of the fimE gene (which mediates an "on" to "off"configuration of the invertible DNA fragment).

A clear indication of a more frequent activation of the fimA promoterand ensuing killing of the host in the case of the pLP4+pPKL100combination appeared from a generation time of 140 minutes for thecorresponding host as compared to 110 minutes for hosts containing thepLP6+pPKL100 combination (Table 3). Furthermore, the former showed thepresence of approximately 12% of ghost cells and the latter to havevirtually none, when the cells were inspected microscopically.

                  TABLE 3                                                         ______________________________________                                                      Generation time                                                 Plasmids      (population doubling time)                                      ______________________________________                                        pPKL100 + pLP4                                                                              140 min.                                                        pPKL100 + pLP5                                                                              120 min.                                                        pPKL100 + pLP6                                                                              110 min.                                                        ______________________________________                                    

Deposit of microorganisms

Pursuant to the provisions of the Budapest Treaty on the InternationalRecognition of Microorganisms for the Purposes of Patent Procedures,samples of the following microorganisms were deposited on Mar. 25, 1987,in Deutsche Sammlung von Mikroorganismen, Grisebachstrasse 8, 3400Gottingen, Federal Republic of Germany:

    ______________________________________                                        Strain                  Accession No.                                         ______________________________________                                        B. subtilis BD170 (pLK26)                                                                             DSM 4037                                              E. coli HB101 (pNL7)    DSM 4034                                              E. coli K-12 MC1000 (pKG345)                                                                          DSM 4036                                              E. coli K-12 MC1000 (pPKL100)                                                                         DSM 4035                                              E. coli K-12 MC1000 (pPKL100 + pLP4)                                                                  DSM 4031                                              E. coli K-12 MC1000 (pPKL100 + pLP5)                                                                  DSM 4032                                              E. coli K-12 MC1000 (pPKL100 + pLP6)                                                                  DSM 4033                                              ______________________________________                                    

We claim:
 1. A recombinant replicon which comprises a first gene, whoseexpression results in the formation of a toxic product which has a toxiceffect on Enterobacteriaceae cells in which said replicon can replicate,and an invertible promoter which regulates the expression of said firstgene, whereby, when said replicon is introduced into hostEnterobacteriaceae cells in which said promoter is functional, undersuitable conditions of expression said invertible promoter is repeatedlyinverted, leading to stochastic expression of the first gene and toformation of said toxic product, thereby stochastically limiting thelife of said replicon-bearing host cell, where said first gene comprisesa coding sequence which encodes a polypeptide selected from the groupconsisting of R1 Hok, F Hok and the polypeptide encoded by relB-orf3. 2.The replicon of claim 1 wherein the invertible promoter is the promoterof the fimA gene or a functional homologue thereof.
 3. The replicon ofclaim 1 wherein the invertible promoter is operably linked to said firstgene, but not natively associated therewith.
 4. A transformed bacterialcell population comprising Enterobacteriaciae cells harbouring thereplicons of claim 1, said replicons being capable of replicating insaid cells, said invertible promoter being functional in said cells, theexpression of said first gene having a toxic effect on said cells. 5.The cell population of claim 4 where the first gene is the hok gene. 6.The cell population of claim 4 where the cells are E. coli cells.
 7. Thecell population of claim 4 wherein the invertible promoter is thepromoter of the fimA gene.
 8. The transformed cell population of claim 4wherein said replicon further comprises a gene expressing a fusionprotein, said fusion protein comprising an outer surface protein of saidcell and an epitope of interest not native to said cell, whereby saidfusion protein serves as a means for transporting the epitope, whenexpressed, to the outer surface of the cell.
 9. A method ofstochastically limiting a Enterobacteriaceae cell population whichcomprises transforming the cells of said cell population with a repliconaccording to claim 1, said replicon being capable of replicating in saidcells, the expression of said first gene having a toxic effect on saidcells, the frequency of inversion of said invertible promoter in saidcells being such as to stochastically limit the growth of said cellpopulation.
 10. The method of claim 9 wherein the invertible promoter isa fimA promoter.
 11. The method of claim 9, wherein said toxic effect issuch that if said promoter remained "on", at least about 99.9% of thecells would be killed.
 12. The method of claim 9 wherein said toxiceffect is such that at least 99.9% of the cells are killed in one hourif said promoter remains "on".
 13. The method of claim 9 wherein theinvertible promoter has substantially the same inversion frequency asthe fimA promoter.
 14. The method of claim 9 wherein one of themanifestations of said toxic effect is the transformation of at leastsome of the bacterial cells into ghost cells.
 15. The replicon of claim1, said promoter having a phase switch responsive to "on" and "off" geneproducts, being caused to transcribe said first gene by an "on" geneproduct and caused not to transcribe said first gene by an "off" geneproduct, whereby the frequency of expression of said first gene may becontrolled by modulating the relative levels of expression of said "on"and "off" gene products.
 16. The replicon of claim 15, furthercomprising a gene encoding an "on" gene product which directs the phaseswitch of said invertible promoter into the "on" position.
 17. Thereplicon of claim 16 wherein the "on" product encoding gene is the fimBgene or a functional homologue thereof.
 18. The replicon of claim 15,further comprising a gene encoding an "off" gene product which directsthe phase switch of said invertible promoter into the "off" position.19. The replicon of claim 18 wherein the "off" product encoding gene isthe fimE gene or a functional homologue thereof.
 20. A transformedbacterial cell of a first kind of cell, said transformed cellcomprising;(a) a recombinant extrachromosomal replicon comprising afirst gene expressed under the control of a regulatable promoter, whoseexpression results in formation of a toxic product which has a lethaleffect on both cells of said first kind and on bacterial cells of asecond kind with which said first kind of cells is capable of naturallyexchanging genetic information, the promoter being functional in bothkinds of cells; and (b) a second replicon comprising a second gene whichencodes a gene product which inhibits expression of such first gene,said second gene being lacking in cells of said second kind, said firstand second kinds of cells both being selected from the group consistingof Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, wheresaid first gene comprises a coding sequence which encodes a polypeptideselected from the group consistinq of R1 Hok, F Hok and the polypeptideencoded by relB - orf3.
 21. The transformed cell of claim 20 in whichthe second replicon is a recombinant chromosomal replicon.
 22. Thetransformed cell of claim 21, wherein the second gene encodes arepressor polypeptide which inhibits transcription of said first gene.23. The transformed cell of claim 22, wherein the second gene encodestrp repressor and the first gene is operably linked to a trp promoterincluding the operator site for trp repressor.
 24. A method ofcontaining an extrachromosomal recombinant replicon to a first kind ofbacterial cells, where said replicon could be naturally transferred to asecond kind of bacterial cells, which comprises providing on therecombinant extrachromosomal replicon a first gene, expressed under thecontrol of a regulatable promoter which is functional in both kinds ofcells, whose expression results in formation of a toxic product whichhas a lethal effect on the first and second kind of cells, said firstkind of cells having or being modified to have a second repliconcomprising a second gene which encodes a gene product which inhibits theexpression of said first gene and thereby protects said first kind ofcells, said second gene being lacking in said second kind of cells,whereby if a cell of the second kind receives said extrachromosomalrecombinant replicon said first gene is expressed and said toxic productis formed, which has a toxic effect thereon, said first and second kindsof cells both being selected from the group consisting ofEnterobacteriaceae, Pseudomonadaceae, and Bacillaceae cells, where saidfirst gene comprises a coding sequence which encodes a polypeptideselected from the group consisting of R1 Hok, F Hok and the polypeptideencoded by relB-orf3.
 25. The method of claim 24 which said secondreplicon is a chromosomal replicon.
 26. The method of claim 25, whereinsaid second gene encodes a repressor polypeptide which inhibitstranscription of said first gene.
 27. The method of claim 26, whereinsaid repressor polypeptide is the trp repressor and said first gene isoperably linked to a trp promoter including the operator site for saidtrp repressor.
 28. The method of claims 25, wherein the chromosomalreplicon is recombinant and the second gene is not natively associatedwith said chromosomal replicon.
 29. The method of claim 24 wherein saidlethal effect is such that at least 99.9% of the cells are killed in onehour if not protected by said inhibitory gene product.
 30. The method ofclaim 24 wherein the cells are gram-negative bacterial cells.
 31. Themethod of claim 24 wherein one of the manifestations of said toxiceffect is the transformation of at least some of the bacterial cellsinto ghost cells.
 32. The method of claim 24 wherein the cells areEnterobacteriaceae cells.
 33. A method of biologically containingbacterial cells growing in a first, controllable environment, whichcells could escape to a second and physically distinct environment,which comprises:(a) providing in said cells a recombinant replicon, saidreplicon comprising a first gene, expressed under the control of aregulatable promoter functional in said cells, whose expression resultsin formation of a toxic product which has a toxic effect on said cells,said cells natively containing or being modified to contain a secondgene whose product inhibits expression of said first gene, saidinhibition being regulatable by an environmental factor, the level ofsaid environmental factor in the second environment being such that saidfirst gene is expressed and said toxic product is formed, whereby cellsin the second environment which harbor said replicon are killed, and (b)manipulating the first environment such that the level of theenvironmental factor therein is such that expression of said first geneis inhibited, whereby cells bearing said replicon are able to grow insaid first environment but not in said second environment, the toxiceffect being such that at least 99.9% of the cells are killed in saidsecond environment, which but for said effect is an environment in whichsaid cells can grow, where said bacterial cells are selected from thegroup consisting of Enterobacteriaceae, Pseudomonadaceae and Bacillaceaecells, where said first gene comprises a coding sequence which encodes apolypeptide selected from the group consisting of R1 Hok, F Hok and thepolypeptide encoded by relB-orf3.
 34. The method of claim 33, whereinthe environmental factor is temperature.
 35. The method of claim 33,wherein the environmental factor is the concentration of a regulatorchemical.
 36. The method of claim 35, wherein the environmental factoris the concentration of tryptophan, the promoter is the trp promoter,the second gene is the trp repressor gene, and sufficient tryptophan isadded to said first environment to provide an inhibitory concentrationthereof.
 37. The method of claim 33, wherein said second gene expressesa repressor polypeptide which inhibits transcription of said first gene.38. The method of claim 33, wherein the second gene is located on thesame replicon as said first gene.
 39. The method of claim 33, whereinthe second gene is located on a chromosomal replicon and the first geneon an extrachromosomal replicon.
 40. The method of claim 33, wherein thetoxic effect is such that at least 99.9% of the cells are killed withinone hour of escape into the second environment.
 41. The method of claim33 wherein the cells are gram-negative bacterial cells.
 42. The methodof claim 33 wherein one of the manifestations of said toxic effect isthe transformation of at least some of the bacterial cells into ghostcells.
 43. The method of claim 33 wherein the cells areEnterobacteriaceae cells.
 44. A method of biologically containingbacterial cells growing in an initial environment subject to physical orchemical change resulting in a changed environment, so that said cellsare able to grow in the initial environment but not in the changedenvironment, which comprisesproviding in said cells a recombinantreplicon, said replicon comprising a first gene, expressed under thecontrol of a regulatable promoter functional in said cells, whoseexpression results in formation of a toxic product which has a toxiceffect on said cells, said cells natively containing or being modifiedto contain an inhibitory gene whose product inhibits expression of saidfirst gene, said inhibition being regulatable by an environmentalfactor, the level of said environmental factor in said changedenvironment being such that said first gene is expressed and said toxicproduct is formed, whereby said cells in said changed environment whichharbor said replicon are killed, the level of the environmental factorin said initial environment being such that expression of said firstgene is inhibited, whereby cells bearing said replicon are able to growin the initial environment but not in the changed environment, the toxiceffect being such that at least 99.9% of the cells are killed in saidchanged environment, which but for said effect is an environment inwhich said cells can grow, said cells being selected from the groupconsisting of Enterobacteriaceae, Pseudomonadaceae, and Bacillaceaecells,where said first gene comprises a coding sequence which encodes apolypeptide selected from the group consisting of R1 Hok, F Hok and thepolypeptide encoded by relB-orf3.
 45. The method of claim 44 wherein theenvironmental factor is the concentration of a chemical in theenvironment.
 46. The method of claim 44, wherein the toxic effect issuch that at least 99.9% of the cells are killed within one hour of saidphysical or chemical change in the environment.
 47. The method of claim44 wherein the cells are gram-negative bacterial cells.
 48. The methodof claim 44 wherein one of the manifestations of said toxic effect isthe transformation of at least some of the bacterial cells into ghostcells.
 49. The method of claim 44 wherein the cells areEnterobacteriaceae cells.